Recombinant neprosin polypeptide and expression method

Abstract

The present disclosure relates to methods of expressing recombinant neprosin polypeptides, host cells and expression vectors for expressing recombinant neprosin polypeptides, and methods of using the expressed neprosin polypeptides for therapeutic and other purposes.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Application No. 63 / 340,411, filed on May 10, 2022, and U.S. Provisional Application No. 63 / 356,181, filed on June 28, 2022, under 35 USC § 119(e), the contents of all of which are incorporated herein by reference. Reference to a sequence listing, table or computer program

[0002] The sequence listing filed concurrently herewith with the file name CX7-229WO3_ST26.txt, created on May 10, 2023, and having a file size of 187,597 bytes, is part of this specification and is incorporated herein by reference. Technical Field

[0003] The present disclosure relates to recombinant neprosin polypeptides, polynucleotides encoding recombinant neprosin polypeptides, and methods of expressing recombinant polynucleotides. Also provided are uses of recombinant neprosin polypeptides for therapeutic and other purposes. background

[0004] Celiac disease is a chronic autoimmune disorder that usually affects the small intestine. The symptoms of the disease are caused by reactions to gluten and other storage proteins (e.g., serpin, purinin, etc.) in edible cereal products. Clinically, the disease can be detected in part by quantification of specific antibodies to gluten and tissue transglutaminase (tTG). The autoimmune response leads to atrophy of the villi of the small intestinal mucosa accompanied by crypt hyperplasia and mucosal inflammation. The symptoms of celiac disease can vary from person to person and may include one or more of the following: fatigue, chronic diarrhea, constipation, malabsorption of nutrients, weight loss, abdominal distension, anemia, and a significantly increased risk of osteoporosis and intestinal malignancies (e.g., lymphoma and epithelial cancer).

[0005] Celiac disease is also a risk factor for other diseases. For example, autism is linked to celiac disease, and a gluten-free diet may help alleviate some symptoms of autism. Similarly, it is believed that when gluten is removed from the diet of some people with attention deficit hyperactivity disorder, they exhibit fewer symptoms. Other conditions that may benefit from eliminating dietary gluten include rheumatoid arthritis and fibromyalgia.

[0006] Treatment of gluten intolerance, particularly celiac disease, usually involves a lifelong strict gluten-free diet. However, a gluten-free diet is inconvenient, restrictive, and gluten is difficult to avoid. Proteases that act on gluten have been proposed as potential therapeutic agents for reducing the inflammatory effects of gluten and mitigating its role in celiac disease. One such protease is the neprosin class of prolyl endopeptidases (PEPs) found in insectivorous tropical pitcher plants of the genus Nepenthes. Neprosin can hydrolyze proline-rich gliadins, which, along with glutenin, are the two main proteins of gluten and are associated with the initiation of celiac disease in susceptible individuals. Small amounts of neprosin are present in Nepenthes pitcher plants, and therefore other sources of neprosin are needed to evaluate proteases in therapeutic applications. Overview

[0007] The present disclosure provides recombinant neprosin polypeptides, methods of expressing recombinant neprosin polypeptides, which result in efficient expression of neprosin, particularly in soluble form, and uses of recombinant neprosin polypeptides in treating diseases or conditions associated with gluten intolerance, such as celiac disease.

[0008] In one aspect, the disclosure provides a recombinant neprosin polypeptide comprising an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence corresponding to a prepropolypeptide, propolypeptide, mature polypeptide or polypeptide of SEQ ID NO: 1, wherein the amino acid sequence comprises a substitution with an amino acid other than asparagine (N) at at least amino acid position 68, 145, 152 or 253, or a combination thereof, and the equivalent thereof, wherein the amino acid position is relative to the reference sequence corresponding to SEQ ID NO: 1.

[0009] In some embodiments, the recombinant neprosin polypeptide comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence corresponding to residues 105 to 356 of SEQ ID NO: 8, or to a reference sequence corresponding to SEQ ID NO: 8, wherein the amino acid sequence comprises a substitution with an amino acid other than asparagine (N) at at least amino acid position 44, 121, 128 or 229, or a combination thereof, wherein the amino acid positions are relative to SEQ ID NO: 8.

[0010] In some embodiments, the amino acid sequence of the recombinant neprosin polypeptide comprises a substitution with an amino acid other than asparagine (N) at at least amino acid position 44. In some embodiments, the amino acid sequence of the recombinant neprosin polypeptide comprises SEQ ID NO: 10.

[0011] In some embodiments, the amino acid sequence of the recombinant neprosin polypeptide comprises a substitution with an amino acid other than asparagine (N) at at least amino acid position 121. In some embodiments, the amino acid sequence of the recombinant neprosin polypeptide comprises residues 105 to 356 of SEQ ID NO: 12, or comprises SEQ ID NO: 12.

[0012] In some embodiments, the amino acid sequence of the recombinant neprosin polypeptide comprises a substitution with an amino acid other than asparagine (N) at at least amino acid position 128. In some embodiments, the amino acid sequence of the recombinant neprosin polypeptide comprises residues 105 to 356 of SEQ ID NO: 14, or comprises SEQ ID NO: 14.

[0013] In some embodiments, the amino acid sequence of the recombinant neprosin polypeptide comprises a substitution with an amino acid other than asparagine (N) at at least amino acid position 229. In some embodiments, the amino acid sequence of the recombinant neprosin polypeptide comprises residues 105 to 356 of SEQ ID NO: 16, or comprises SEQ ID NO: 16.

[0014] In some embodiments, the amino acid sequence of the recombinant neprosin polypeptide comprises a substitution with an amino acid other than asparagine (N) at at least amino acid positions 121 and 229. In some embodiments, the amino acid sequence of the recombinant neprosin polypeptide comprises residues 105 to 356 of SEQ ID NO: 18, or comprises SEQ ID NO: 18.

[0015] In some embodiments, the amino acid sequence of the recombinant neprosin polypeptide comprises substitutions with amino acids other than asparagine (N) at at least amino acid positions 44, 128, and 229. In some embodiments, the amino acid sequence of the recombinant neprosin polypeptide comprises residues 105 to 356 of SEQ ID NO: 20, or comprises SEQ ID NO: 20.

[0016] In some embodiments, the amino acid sequence of the recombinant neprosin polypeptide comprises substitutions with amino acids other than asparagine (N) at at least amino acid positions 44, 121, 128, and 229. In some embodiments, the amino acid sequence of the recombinant neprosin polypeptide comprises residues 105 to 356 of SEQ ID NO: 22, or comprises SEQ ID NO: 22.

[0017] In some embodiments, the amino acid sequence of the recombinant neprosin polypeptide comprises a substitution with an amino acid other than asparagine (N) at at least amino acid positions 44 and 229. In some embodiments, the amino acid sequence of the recombinant neprosin polypeptide comprises residues 105 to 356 of SEQ ID NO: 32, or comprises SEQ ID NO: 32.

[0018] In some embodiments, the amino acid sequence of the recombinant neprosin polypeptide further comprises a signal sequence. In some embodiments, the signal sequence is a heterologous signal sequence. In some embodiments, the signal sequence is functional in mammalian cells, insect cells, or fungal cells.

[0019] In some embodiments, the recombinant neprosin polypeptide comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence corresponding to residues 105 to 356 of SEQ ID NO: 8, or to a reference sequence corresponding to SEQ ID NO: 8, wherein the amino acid sequence further comprises a signal sequence that is functional in a mammalian cell, an insect cell, or a fungal cell. In some embodiments, the amino acid sequence of the recombinant neprosin polypeptide comprises residues 105 to 356 of SEQ ID NO: 8, or comprises SEQ ID NO: 8.

[0020] In some embodiments, the recombinant neprosin polypeptide is glycosylated. In some embodiments, the recombinant neprosin polypeptide is not glycosylated at least at amino acid position 44, 121, 128, or 229, wherein the amino acid positions are relative to SEQ ID NO: 8. In some embodiments, the recombinant neprosin polypeptide is glycosylated, but is not glycosylated at least at amino acid position 44, 121, 128, or 229, wherein the amino acid positions are relative to SEQ ID NO: 8. In some embodiments, the recombinant neprosin polypeptide is not glycosylated.

[0021] In another aspect, the disclosure provides a recombinant polynucleotide comprising a polynucleotide sequence encoding a recombinant neprosin polypeptide described herein. In some embodiments, the polynucleotide sequence is codon-optimized, preferably codon-optimized for expression in mammalian cells, insect cells, or fungal cells.

[0022] In another aspect, the disclosure provides an expression vector comprising a recombinant polynucleotide encoding a recombinant neprosin polypeptide. In some embodiments, the expression vector comprises a control sequence such as a promoter preferably for expressing a recombinant neprosin polypeptide in mammalian cells, insect cells or fungal cells.

[0023] In another aspect, a host cell comprising an expression vector is also provided. In some embodiments, the host cell is a eukaryotic cell, preferably a mammalian cell, an insect cell or a fungal cell.

[0024] In some embodiments, the host cell is a mammalian cell comprising an expression vector comprising a polynucleotide sequence encoding a recombinant neprosin polypeptide. In some embodiments, the mammalian cell is a human cell or a rodent cell. In some embodiments, the mammalian cell is Expi293, HeLa, U2OS, A549, HT1080, CAD, P19, NIH 3T3, L929, Hek 293, 293F, 293E, 293T, COS, Vero, NS0 cells, Sp2 / 0 cells, DUKX-X11, MCF-7, Y79, SO-Rb50, Hep G2, J558L or CHO cells. In some embodiments, the polynucleotide sequence encoding a recombinant neprosin polypeptide is codon-optimized for expression in mammalian cells.

[0025] In some embodiments, the host cell comprises an insect cell comprising an expression vector comprising a polynucleotide sequence encoding a recombinant neprosin polypeptide. In some embodiments, the insect cell is a lepidopteran or dipteran insect cell. In some embodiments, the insect cell is a Sf9 cell, a Sf21 cell, a Schneider 2 or a High Five cell. In some embodiments, the polynucleotide sequence encoding a recombinant neprosin polypeptide is codon-optimized for expression in insect cells.

[0026] In some embodiments, the host cell includes a fungal cell, which includes an expression vector, which includes a polynucleotide sequence encoding a recombinant neprosin polypeptide. In some embodiments, the fungal cell is a filamentous fungal cell. In some embodiments, the fungal cell is a yeast cell. In some embodiments, the fungal cell is a Pichia, Saccharomyces, Yarrowia, Kluyveromyces, Aspergillus, Trichoderma, Neurospora, Mucor, Penicillium, Trichoderma or Myceliophthora. In some embodiments, the polynucleotide sequence encoding the recombinant neprosin polypeptide is codon optimized for expression in fungal cells.

[0027] In another aspect, the present disclosure provides a method for expressing a recombinant neprosin polypeptide, the method comprising culturing a host cell comprising an expression vector comprising a polynucleotide sequence encoding a recombinant neprosin polypeptide under suitable conditions, such that the recombinant neprosin polypeptide is expressed.

[0028] In some embodiments of the method, the expressed recombinant neprosin polypeptide is a mature neprosin polypeptide. In some embodiments, the expressed recombinant neprosin polypeptide is a propolypeptide / proenzyme form of a neprosin polypeptide. In some embodiments, the expressed recombinant neprosin polypeptide is a prepropolypeptide / preenzyme form of a neprosin polypeptide. In some embodiments, the expressed recombinant neprosin polypeptide is secreted. In some embodiments, the recombinant neprosin polypeptide is expressed in a soluble form.

[0029] In some embodiments, the expressed recombinant neprosin polypeptide is glycosylated. In some embodiments, the expressed recombinant neprosin polypeptide is not glycosylated at least at amino acid position 44, 121, 128, or 229, wherein the amino acid positions are relative to SEQ ID NO: 8. In some embodiments, the expressed recombinant neprosin polypeptide is glycosylated, but is not glycosylated at least at amino acid position 44, 121, 128, or 229, wherein the amino acid positions are relative to SEQ ID NO: 8. In some embodiments, the expressed recombinant neprosin polypeptide is non-glycosylated.

[0030] In another aspect, the disclosure provides a method for preparing a mature neprosin polypeptide, particularly under in vitro conditions, the method comprising treating a full-length neprosin polypeptide or a pre-propolypeptide or propolypeptide of a neprosin polypeptide under acidic conditions suitable for forming a mature neprosin polypeptide. In some embodiments, the acidic conditions are a pH of about 2 to 6, or a pH of about 2 to about 4.5, preferably a pH of about 2 to about 3. In some embodiments, the mature neprosin polypeptide is prepared by proteolytic cleavage. In some embodiments, the mature neprosin prepared by the method has proteolytic activity.

[0031] In another aspect, the present disclosure provides a pharmaceutical composition comprising a recombinant neprosin polypeptide or a pro-polypeptide or mature polypeptide form of a recombinant neprosin polypeptide. In some embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable excipient and / or carrier.

[0032] In another aspect, the disclosure provides a method of treating or preventing symptoms of a disease or condition associated with gluten intolerance, the method comprising administering to a subject in need thereof an effective amount of a recombinant neprosin polypeptide described herein or a pharmaceutical composition thereof. In some embodiments, the recombinant neprosin polypeptide or a pharmaceutical composition thereof is administered before, simultaneously with, or after consumption of a gluten-containing food. In some embodiments, the disease or condition is associated with gluten intolerance in celiac disease. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1A Depicted are the amino acid sequence of the neprosin polypeptide of SEQ ID NO: 1 and proposed signal peptide, activation peptide, domains of mature neprosin, and glycosylation sites at amino acid positions 68, 145, 152, and 253 relative to SEQ ID NO: 1. Figure 1B The amino acid sequence of the neprosin propolypeptide of SEQ ID NO: 8 and the pro- and mature neprosin protease regions of the neprosin protease are depicted. Glycosylation sites at amino acid positions 44, 121, 128 and 229 relative to SEQ ID NO: 8 are also shown, which are equivalent to amino acid positions 68, 145, 152 and 253 of SEQ ID NO: 1.

[0034] Figure 2 Photographs showing SDS PAGE analysis of the formation of activated neprosin protease polypeptide at pH 2.5 for neprosin expressed in CHO cells (Fig. A) and neprosin expressed in Expi293 cells. The activated neprosin polypeptide appears as a polypeptide of approximately 25.5 kDa in the SDS PAGE system used.

[0035] Figure 3 Photographs of SDS PAGE analysis of the formation of proteolytically active mature neprosin polypeptides under (i) different pH, (ii) unbuffered simulated gastric fluid (SGF) (pH 7) or (iii) unbuffered SGF + pepsin conditions as analyzed by SDS PAGE are shown. Panel A is an SDS-PAGE of neprosin protein dialyzed in buffers of different pH, SGF or SGF + pepsin. Panel B is an SDS-PAGE of neprosin protein after 5 h dialysis.

[0036] Figure 4A graph comparing the proteolytic activity of mature activated neprosin polypeptide and neprosin polypeptide prepared from cells expressing mSP-His-Nprl towards a gliadin substrate is provided.

[0037] Figure 5 Provided is a photograph of SDS PAGE analysis of recombinant neprosin propolypeptides expressed in Pichia cells by wild-type (WT) construct (SEQ ID NO: 56), N216A mutant construct (SEQ ID NO: 60) and N216A / N324A mutant construct (SEQ ID NO: 66). WT, N216A and N216A / N324A neprosin propolypeptide forms appear as heterogeneous mixtures of glycosylated polypeptides, as evidenced by a smear of about 60 kDa on the SDS-PAGE gel. The smaller molecular weight band of about 29 kDa corresponds to an activated form of each of these proteins, which is produced during expression and purification. Amino acid positions 216 and 324 of SEQID NO: 60 and SEQ ID NO: 66 are equivalent to amino acid positions 121 and 229 of SEQ ID NO: 8, respectively.

[0038] Figure 6 Provided is a photograph of SDS-PAGE analysis of the formation of activated neprosin protease polypeptides at pH 2.5 for WT constructs (SEQ ID NO: 56), N216A mutant constructs (SEQ ID NO: 60) and N216A / N324A mutant constructs (SEQ ID NO: 66) expressed in Pichia cells. Activated WT neprosin is expressed as two glycosylated polypeptides of approximately 29 kDa and 35 kDa. Activated N216A neprosin and N216A / N324A neprosin are expressed as monoglycosylated species of approximately 29 kDa.

[0039] Figure 7 A graph is provided of the proteolytic activity of activated neprosin Npr1 prepared from Npr1 expressed in Pichia pastoris cells against gliadin substrates in buffered SGF conditions (pH 3, 0.8 mg / mL pepsin) compared to activated Npr1 prepared from Npr1 expressed in Chinese hamster ovary (CHO) cells or no added neprosin enzyme. The activity of pepsin included in SGF against gliadin substrates is responsible for the reduced gliadin levels observed in the "no enzyme" samples.

[0040] Figure 8A graph is provided of the proteolytic activity of activated N216A Nprl (SEQ ID NO: 60) and N216A / N324A Nprl (SEQ ID NO: 66) prepared from recombinant neprosin polypeptide expressed in Pichia pastoris cells, compared to activated WT Nprl prepared from Nprl expressed in Pichia pastoris cells or no added neprosin enzyme, on gliadin substrates in buffered SGF conditions (pH 3, 0.8 mg / mL pepsin). The activity of pepsin contained in SGF on gliadin substrates is responsible for the reduced gliadin levels observed in the "no enzyme" samples.

[0041] Fig. 9 A graph is provided of the dose-dependent proteolytic activity of activated WT Npr1 prepared from Npr1 expressed in CHO cells on gliadin substrates in mouse stomach. Data are shown as mean ± standard deviation. The statistical test applied was Dunnett's T3 multiple comparison test with: *p≤0.05, **p≤0.01, ***p≤0.001 and ****p≤0.0001.

[0042] Fig.10 Figures showing the level of T cell activation in peripheral blood mononuclear cells (PBMCs) collected from five human patients with celiac disease and challenged with gluten, treated or untreated with activated WT Npr1 prepared from Npr1 expressed in CHO cells. Activated WT Npr1 activity was tested in 15 min and 60 min gastric digestion. Positive control data were digested and deamidated gliadin (D-gliadin) with buffer (60 min gastric digestion) as vehicle control. Data are shown as mean ± standard deviation. The statistical test applied was the Friedman test, with: *p≤0.05, **p≤0.01 and ***p≤0.001. Details

[0043] The present disclosure provides recombinant neprosin polypeptides, recombinant polynucleotides encoding recombinant neprosin polypeptides, host cells comprising the recombinant polynucleotides, and methods of expressing the recombinant neprosin polypeptides using host cells. The present disclosure also provides recombinant neprosin polypeptides prepared from host cells, particularly recombinant neprosin polypeptides in soluble form, and methods for preparing proteolytically active forms of recombinant neprosin for obtaining therapeutic efficacy and uses thereof in treating gluten intolerance. Abbreviations and definitions

[0044] Unless otherwise defined, all technical and scientific terms used herein generally have the same meaning as those generally understood by those of ordinary skill in the art to which the present invention belongs. Typically, the nomenclature used herein and the laboratory procedures for cell culture, molecular genetics, microbiology, organic chemistry, analytical chemistry, and nucleic acid chemistry described below are those well known in the art and commonly employed. Such techniques are well known and described in many textbooks and reference works well known to those skilled in the art. Standard techniques or modifications thereof are used for chemical synthesis and chemical analysis.

[0045] Although any suitable method and material similar or equal to those described herein can be used in the practice of the present invention, some methods and materials are described herein.It should be understood that the present invention is not limited to the described ad hoc method, scheme and reagent, because these can change depending on the situation that those skilled in the art use it.Therefore, the following terms of definition are more fully described by reference to the application as a whole.All patents, patent applications, articles and publications mentioned herein above and below are hereby expressly incorporated herein by reference.

[0046] Furthermore, as used herein, the singular "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0047] As used herein, the term "comprising" and its cognates are used in their inclusive sense (ie, equivalent to the term "including" and its corresponding cognates).

[0048] It will be further understood that where the description of embodiments uses the term "comprising" and its cognates, embodiments may also be described using the language "consisting essentially of" or "consisting of.

[0049] Numerical ranges include the numbers defining the ranges. Therefore, each numerical range disclosed herein is intended to include each narrower numerical range falling within these broader numerical ranges, as if these narrower numerical ranges were all clearly written herein. It is also intended that each maximum (or minimum) numerical limit disclosed herein includes each lower (or higher) numerical limit, as if these lower (or higher) numerical limits are clearly written herein.

[0050] As used herein, the term "about" means an acceptable error for a particular value. In some cases, "about" means within 0.05%, 0.5%, 1.0%, or 2.0% of a given value range. In some cases, "about" means within 1, 2, 3, or 4 standard deviations of a given value.

[0051] In addition, the titles provided herein are not limitations of the various aspects or embodiments of the present invention that may be possessed by reference to the present application as a whole. Therefore, the terms defined immediately below are more fully defined by reference to the present application as a whole. Nevertheless, for ease of understanding of the present invention, many terms are defined as follows.

[0052] "EC" numbers refer to the enzyme nomenclature of the International Union of Biochemistry and Molecular Biology (NC-IUBMB). The IUBMB biochemical classification is a numerical classification system for enzymes based on the chemical reactions that they catalyze.

[0053] "ATCC" refers to the American Type Culture Collection, whose biological collection includes genes and strains.

[0054] "NCBI" refers to the National Center for Biological Information and the sequence databases provided therein.

[0055] "Polynucleotide" is used herein to refer to a polymer comprising at least two nucleotides, wherein the nucleotide is a deoxyribonucleotide or a ribonucleotide. The abbreviations for genetically encoded nucleosides are conventional and are as follows: adenosine (A); guanosine (G); cytidine (C); thymidine (T); and uridine (U). Unless otherwise specified, the abbreviated nucleosides can be ribonucleosides or 2'-deoxyribonucleosides. Nucleosides can be designated as ribonucleosides or 2'-deoxyribonucleosides on an individual or overall basis. When a nucleic acid sequence is presented as a string of single-letter abbreviations, the sequence is presented in a 5' to 3' direction according to conventional practice, and the phosphate is not shown.

[0056] "Duplex" and "ds" refer to a double-stranded nucleic acid (e.g., DNA) molecule composed of two single-stranded polynucleotides that are complementary in sequence (A pairs with T, C pairs with G), arranged in an anti-parallel 5' to 3' direction, and held together by hydrogen bonds between the nucleobases (i.e., adenine [A], guanine [G], cytosine [C], and thymine [T]).

[0057] "Protein", "polypeptide" and "peptide" are used interchangeably to refer to a polymer of at least two amino acids covalently linked by amide bonds, regardless of length or post-translational modifications (e.g., glycosylation or phosphorylation). Unless otherwise indicated, amino acid sequences are written from left to right in the direction of amino to carboxyl.

[0058] "Amino acids" are referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. The abbreviations used for the genetically encoded amino acids are conventional and are as follows: alanine (Ala or A), arginine (Arg or R), asparagine (Asn or N), aspartic acid (Asp or D), cysteine ​​(Cys or C), glutamic acid (Glu or E), glutamine (Gln or Q), glycine (Gly or G), histidine (His or H), isoleucine (Ile or I), leucine (Leu or L), lysine (Lys or K), methionine (Met or M), phenylalanine (Phe or F), proline (Pro or P), serine (Ser or S), threonine (Thr or T), tryptophan (Trp or W), tyrosine (Tyr or Y), and valine (Val or V). When three-letter abbreviations are used, unless explicitly preceded by "L" or "D" or it is obvious from the context in which the abbreviation is used, the amino acid can be in the L-configuration or the D-configuration about the α-carbon (Cα). For example, "Ala" represents alanine without specifying the configuration about the α-carbon, while "D-Ala" and "L-Ala" represent D-alanine and L-alanine, respectively. When single-letter abbreviations are used, capital letters represent amino acids in the L-configuration about the α-carbon, and lowercase letters represent amino acids in the D-configuration about the α-carbon. For example, "A" represents L-alanine, and "a" represents D-alanine. When a polypeptide sequence is presented as a string of single-letter or three-letter abbreviations (or a mixture thereof), the sequence is presented in the amino (N) to carboxyl (C) direction according to common convention.

[0059] "Fusion protein" and "chimeric protein" and "chimera" refer to hybrid proteins produced by connecting two or more genes that originally encoded separate proteins. In some embodiments, the fusion protein is produced by recombinant technology (e.g., molecular biology techniques known in the art).

[0060] "Neprosin" refers to a prolyl endoprotease originally isolated from the secretions of Nepenthes species. Neprosin is known to cleave proteins carboxyl termini to proline with high specificity. The enzyme is active at about pH 2 to about pH 5. As used herein, neprosin includes isoforms, isotypes, homologs, orthologs, variants and recombinant neprosin and salts thereof. Exemplary neprosin polypeptides are disclosed in WO2015192211 and Ting et al., Plant Physiol Biochem., 2022, 183: 23-35, which are incorporated herein by reference.

[0061] "Nepenthesin" refers to an aspartic protease having an Enzyme Commission Number EC 3.4.23.12, and includes all isoforms, isoforms and variants of Nepenthesin, such as Nepenthesin I and Nepenthesin II, Nepenthesin isoforms and recombinant Nepenthesin and salts thereof. Nepenthesin is a plant-derived aspartic protease that can be isolated or concentrated from a variety of plant sources, such as Nepenthes secretions of the genus Nepenthes, commonly known as monkey cups in tropical regions. Nepenthesin is described in International Patent Publications WO2014078935 and WO2014138927, which are incorporated herein by reference in their entirety.

[0062] "Mature protein" or "mature polypeptide" refers to the final processed biological protein or polypeptide or product.

[0063] "Pro-protein", "pro-polypeptide" or "pro-peptide" refers to a precursor protein, polypeptide or peptide that is processed by post-translational modification to form a biologically active protein, polypeptide or peptide. In some embodiments, the post-translational modification is a cleavage reaction to form a protein, polypeptide or peptide. "Pro-enzyme" refers to a precursor polypeptide that is processed by post-translational modification, particularly a cleavage reaction, to form an active enzyme.

[0064] "Pre-pro-protein", "pre-pro-polypeptide" or "pre-pro-peptide" refers to a precursor protein, polypeptide or peptide that includes a signal sequence and can be processed by post-translational modification (particularly a cleavage reaction) to produce a proprotein, propolypeptide or propeptide. Typically, the cleavage reaction removes the signal sequence to produce a proprotein, propolypeptide or propeptide. "Preproenzyme" refers to a precursor protein, polypeptide or peptide that is processed by post-translational modification (particularly a cleavage reaction that removes the signal sequence) to form a zymogen.

[0065] In the context of a protein or polypeptide, "full length" refers to a protein or polypeptide that has not been processed to change the amino acid sequence of the entire protein or polypeptide. For example, a full-length protein is the entire protein encoded in the corresponding mRNA.

[0066] "Engineered," "recombinant," "non-naturally occurring," and "variant" when used with respect to cells, polynucleotides, or polypeptides refer to materials that are or correspond to the native or natural form of that material: have been modified or are identical to a manner not originally found in nature but are produced or derived from synthetic materials and / or through manipulation using recombinant techniques.

[0067] "Wild-type" and "naturally occurring" refer to the form found in nature. For example, a wild-type polypeptide or polynucleotide sequence is a sequence present in an organism, which can be isolated from a natural source, and which has not been intentionally modified by human manipulation.

[0068] "Coding sequence" refers to the portion of a nucleic acid (eg, a gene) that encodes the amino acid sequence of a protein.

[0069] "Percentage (%) of sequence identity" is used herein to refer to a comparison between polynucleotides and polypeptides, and is determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide or polypeptide sequence in the comparison window may include additions or deletions (i.e., gaps) compared to the reference sequence for the optimal alignment of the two sequences. The percentage may be calculated by determining the number of positions where the same nucleic acid base or amino acid residue appears in the two sequences to produce the number of matching positions, dividing the number of matching positions by the total number of positions in the comparison window, and multiplying the result by 100 to produce the percentage of sequence identity. Alternatively, the percentage may be calculated by determining the number of positions where the same nucleic acid base or amino acid residue appears in the two sequences or where one nucleic acid base or amino acid residue is aligned with a gap to produce the number of matching positions, dividing the number of matching positions by the total number of positions in the comparison window, and multiplying the result by 100 to produce the percentage of sequence identity. Those skilled in the art will appreciate that there are many established algorithms that can be used to align two sequences. Optimal alignment of sequences for comparison can be performed by (e.g., by the local homology algorithm of Smith and Waterman; Smith and Waterman, Adv. Appl. Math., 1981, 2:482), by the homology alignment algorithm of Needleman and Wunsch (Needleman and Wunsch, J. Mol. Biol., 1970, 48:443), by the search for similarity method of Pearson and Lipman (Pearson and Lipman, Proc. Natl. Acad. Sci. USA., 1988, 85:2444), by computerized implementations of these algorithms (e.g., GAP, BESTFIT, FASTA, and TFASTA in the GCG Wisconsin software package), or by visual inspection, as is known in the art. Examples of algorithms suitable for determining percentages of sequence identity and sequence similarity include, but are not limited to, BLAST and BLAST 2.0 algorithms (see, e.g., Altschul et al., J. Mol. Biol., 1990, 215:403-410; and Altschul et al., Nucleic Acids Res., 1977, 25:3389-3402). Software for performing BLAST analysis is publicly available through the National Center for Biotechnology Information website.This algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length "W" in the query sequence, which, when aligned with a word of the same length in a database sequence, either match or satisfy some positive-valued threshold score "T". T is called the neighborhood word score threshold (see, Altschul et al., supra). These initial neighborhood word hits are used as seeds for initiating searches to find longer HSPs containing them. The word hits are then extended in both directions along each sequence until the cumulative alignment score cannot be increased. For nucleotide sequences, the cumulative score is calculated using the parameters "M" (reward score for matching residue pairs; always >0) and "N" (penalty score for mismatching residues; always <0). For amino acid sequences, a scoring matrix is ​​used to calculate the cumulative score. Extension of the word hits in each direction ceases when: the cumulative alignment score drops by an amount "X" from its maximum achieved value; the cumulative score reaches zero or below due to the accumulation of one or more negatively scored residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T and X determine the sensitivity and speed of comparison. The BLASTN program (for nucleotide sequences) uses the following as default: word length (W) is 11, expected value (E) is 10, M=5, N=-4 and double-stranded comparison. For amino acid sequences, the BLASTP program uses the following as default: 3 word lengths (W), 10 expected values ​​(E) and BLOSUM62 scoring matrix (see, for example, Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA, 1989, 89:10915). The exemplary determination of sequence alignment and sequence identity % can use the default parameters provided, adopting BESTFIT or GAP programs in GCG Wisconsin software package (Accelrys, Madison WI).

[0070] "Reference sequence" refers to a sequence of a definition used as a basis for sequence comparison. A reference sequence can be a subset of a larger sequence, for example, a segment of a full-length gene or polypeptide sequence. Typically, a reference sequence is a length of at least 20 nucleotides or amino acid residues, a length of at least 25 residues, a length of at least 50 residues, a length of at least 100 residues, or the total length of a nucleic acid or polypeptide. Since two polynucleotides or polypeptides can each (1) comprise a similar sequence (i.e., a part of a complete sequence) between the two sequences, and (2) can also comprise a different sequence between the two sequences, a sequence comparison between two (or more) polynucleotides or polypeptides is typically performed by comparing the sequences of two polynucleotides or polypeptides on a "comparison window" to identify and compare the sequence similarity of a local region. In some embodiments, a "reference sequence" can be based on a primary amino acid sequence, wherein a reference sequence is a sequence that can have one or more changes in the primary sequence.

[0071] "Comparison window" refers to a conceptual segment of at least about 20 consecutive nucleotide positions or amino acid residues, wherein a sequence can be compared to a reference sequence of at least 20 consecutive nucleotides or amino acids, and wherein the portion of the sequence in the comparison window can include 20% or less additions or deletions (i.e., spaces) compared to the reference sequence (which does not include additions or deletions) to obtain the best alignment of the two sequences. The comparison window can be longer than 20 consecutive residues, and optionally includes 30, 40, 50, 100 or longer windows.

[0072] When used in the context of numbering a given amino acid or polynucleotide sequence, "corresponding to," "reference to," and "relative to," refer to the numbering of the residues of a given amino acid or polynucleotide sequence when the given amino acid or polynucleotide sequence is compared to a specified reference sequence. In other words, the residue numbers or residue positions of a given polymer are specified with respect to a reference sequence, rather than by the actual numerical position of the residue within the given amino acid or polynucleotide sequence. For example, a given amino acid sequence, such as the amino acid sequence of a recombinant neprosin polypeptide, can be aligned with a reference sequence by introducing gaps to optimize the matching of residues between the two sequences. In these cases, the numbering of the residues in a given amino acid or polynucleotide sequence is performed with respect to the reference sequence to which it is aligned, despite the presence of gaps.

[0073] "Amino acid difference" and "residue difference" refer to the difference of the amino acid residue at one position of a polypeptide sequence relative to the amino acid residue at the corresponding position in the reference sequence. The position of the amino acid difference is generally referred to as "Xn" herein, where n refers to the corresponding position in the reference sequence based on which the residue difference exists. For example, "the residue difference at position X121 compared to SEQ ID NO: 8" (or "the residue difference at position 121 compared to SEQ ID NO: 8") refers to the difference of the amino acid residue at the polypeptide position corresponding to position 121 of SEQ ID NO: 8. Therefore, if the reference polypeptide of SEQ ID NO: 8 has asparagine at position 121, then "the residue difference at position X121 compared to SEQ ID NO: 8" refers to the amino acid substitution of any residue other than asparagine at the position of the polypeptide corresponding to position 121 of SEQ ID NO: 8. In some cases herein, a specific amino acid residue difference at a position is indicated as "XnY," where "Xn" designates the corresponding residue and position of a reference polypeptide (as described above), and "Y" is the single-letter identifier of the amino acid present in the engineered or recombinant polypeptide (i.e., the residue that differs from the reference polypeptide). In some embodiments, an amino acid difference (e.g., a substitution) is indicated by the abbreviation "nY" without the identifier of the residue in the reference sequence.

[0074] "Conservative amino acid substitutions" refer to replacement of a residue with a different residue having a similar side chain, and thus generally include replacement of an amino acid in a polypeptide with an amino acid from the same or similar amino acid defined class. For example, but not limited to, an amino acid with an aliphatic side chain can be replaced by another aliphatic amino acid (e.g., alanine, valine, leucine, and isoleucine); an amino acid with a hydroxyl side chain can be replaced by another amino acid with a hydroxyl side chain (e.g., serine and threonine); an amino acid with an aromatic side chain can be replaced by another amino acid with an aromatic side chain (e.g., phenylalanine, tyrosine, tryptophan, and histidine); an amino acid with a basic side chain can be replaced by another amino acid with a basic side chain (e.g., lysine and arginine); an amino acid with an acidic side chain can be replaced by another amino acid with an acidic side chain (e.g., aspartic acid or glutamic acid); and / or a hydrophobic or hydrophilic amino acid can be replaced by another hydrophobic or hydrophilic amino acid, respectively.

[0075] "Non-conservative substitutions" refer to substitutions of amino acids in a polypeptide with amino acids having significantly different side chain properties. Non-conservative substitutions may use amino acids between, rather than within, the defined groups and affect: (a) the structure of the peptide backbone in the area of ​​the substitution (e.g., proline for glycine); (b) charge or hydrophobicity; and / or (c) the bulk of the side chain. For example, but not limited to, exemplary non-conservative substitutions may be substitutions of acidic amino acids with basic or aliphatic amino acids; substitutions of aromatic amino acids with small amino acids; and substitutions of hydrophilic amino acids with hydrophobic amino acids.

[0076] "Deletion" refers to the modification of a polypeptide by removing one or more amino acids from a reference polypeptide. Deletion can include the removal of 1 or more amino acids, 2 or more amino acids, 5 or more amino acids, 10 or more amino acids, 15 or more amino acids, or 20 or more amino acids, up to 10% of the total number of amino acids that make up the reference enzyme, or up to 20% of the total number of amino acids, while retaining enzymatic activity and / or retaining the properties of the neprosin polypeptide. Deletion can involve internal portions and / or terminal portions of a polypeptide. In various embodiments, deletion can include continuous segments, or can be discontinuous.

[0077] "Insertion" refers to the modification of a polypeptide by adding one or more amino acids to a reference polypeptide. The insertion may be in an internal portion of a polypeptide, or at the carboxyl or amino terminus. Insertions as used herein include fusion proteins as known in the art. Insertions may be continuous stretches of amino acids, or separated by one or more amino acids in a naturally occurring polypeptide.

[0078] "Functional fragment" and "biologically active fragment" are used interchangeably herein to refer to a polypeptide having an amino-terminal and / or carboxyl-terminal deletion and / or an internal deletion, but wherein the remaining amino acid sequence is identical to the corresponding positions in the sequence to which it is being compared (e.g., a full-length recombinant neprosin polypeptide) and retains substantially all of the activity of the full-length polypeptide.

[0079] "Isolated polypeptide" refers to a polypeptide that is substantially separated from other contaminants (e.g., proteins, lipids, and polynucleotides) that naturally accompany the polypeptide. The term includes polypeptides that have been removed or purified from their naturally occurring environment or expression system (e.g., host cells or in vitro synthesis). Recombinant neprosin polypeptides can be present in cells, in cell culture media, or prepared in various forms, such as lysates or isolated preparations. Therefore, in some embodiments, the recombinant neprosin polypeptides provided herein are isolated polypeptides.

[0080] "Substantially pure polypeptide" refers to a composition in which the polypeptide species is the predominant species present (i.e., it is more abundant than any other individual macromolecular species in the composition on a molar or weight basis), and when the species of interest constitutes at least about 50% of the macromolecular species present on a molar or weight % basis, the composition is generally a substantially purified composition. Typically, a substantially pure neprosin polypeptide composition will constitute about 60% or more, about 70% or more, about 80% or more, about 90% or more, about 95% or more, and about 98% or more of all macromolecular species present in the composition on a molar or weight % basis. In some embodiments, the species of interest is purified to substantial homogeneity (i.e., contaminating species cannot be detected in the composition by conventional detection methods), wherein the composition consists essentially of a single macromolecular species. Solvent species, small molecules (<500 Daltons), and elemental ionic species are not considered macromolecular species. In some embodiments, the isolated recombinant neprosin polypeptide is a substantially pure polypeptide composition.

[0081] "Proteolytic activity" and "proteolysis" are used interchangeably herein and refer to the breakdown of proteins into smaller polypeptides or amino acids. The breakdown of proteins is generally the result of the hydrolysis of peptide bonds by proteases (proteinases). Exemplary proteases include, but are not limited to, neprosin, pitcher protease, pepsin, trypsin, chymotrypsin, elastase; carboxypeptidases A and B and peptidases (e.g., aminopeptidases, dipeptidases, and enteropeptidases).

[0082] "Codon optimization" refers to the change of the codons of a polynucleotide encoding a protein to codons preferentially used in a specific organism, so that the encoded protein is more efficiently expressed in the organism. Although the genetic code is degenerate, because most amino acids are represented by several codons referred to as "synonymous" or "synonymous" codons, it is well known that the codon usage of a specific organism is non-random and biased towards specific codon triplets. This codon usage bias may be higher for a given gene, a gene with a common function or ancestral origin, a highly expressed protein compared to a low copy number protein, and a dense protein coding region of the genome of an organism. In some embodiments, the polynucleotide encoding the neprosin polypeptide is codon optimized for optimized production from a selected host organism for expression.

[0083] "Control sequences" herein refer to all components that are necessary or advantageous for the expression of the polynucleotides and / or polypeptides of the present disclosure. Each control sequence may be native or exogenous to the nucleic acid sequence encoding the polypeptide. Such control sequences include, but are not limited to, leader sequences, polyadenylation sequences, propeptide sequences, promoter sequences, signal peptide sequences, start sequences, and transcription terminators. At a minimum, control sequences include promoters and transcription and translation termination signals. In some embodiments, control sequences are provided with linkers for the purpose of introducing specific restriction sites that facilitate the connection of control sequences to the coding region of the nucleic acid sequence encoding the polypeptide.

[0084] "Operably linked" is defined herein as a configuration in which a control sequence is appropriately placed (i.e., in a functional relationship) relative to a polynucleotide of interest such that the control sequence directs or regulates the expression of the polypeptide encoding the polynucleotide and / or polypeptide of interest.

[0085] "Heterologous" or "recombinant" refers to the relationship between two or more nucleic acid or polypeptide sequences (eg, promoter sequence, signal peptide, terminator sequence, etc.) that are derived from different sources and are not essentially related.

[0086] "Promoter sequence" refers to a nucleic acid sequence that is recognized by a host cell for expression of a polynucleotide of interest (such as a coding sequence). The promoter sequence comprises a transcriptional control sequence that mediates the expression of the polynucleotide of interest. The promoter can be any nucleic acid sequence that exhibits transcriptional activity in a selected host cell, including mutant, truncated and hybrid promoters, and can be obtained from a gene encoding an extracellular polypeptide or an intracellular polypeptide that is homologous or heterologous to the host cell.

[0087] "Vector" refers to a polynucleotide construct for introducing a polynucleotide sequence into a cell. In some embodiments, a vector is an expression vector operably linked to a suitable control sequence capable of achieving expression of a polypeptide encoded in a polynucleotide sequence in a suitable host. In some embodiments, an "expression vector" has a promoter sequence operably linked to a polynucleotide sequence (e.g., a transgene) to drive expression in a host cell, and in some embodiments, further comprises a transcription terminator sequence.

[0088] "Expression" includes any step involved in the production of the polypeptide, including but not limited to transcription, post-transcriptional modification, translation, and post-translational modification. In some embodiments, the term also includes secretion of the polypeptide from the cell.

[0089] "Culturing" refers to the growth of a population of cells, such as host cells, under suitable conditions using any suitable medium (eg, liquid, gel, or solid).

[0090] "Produce" refers to the production of proteins and / or other compounds by a cell. The term is intended to encompass any step involved in the production of a polypeptide, including, but not limited to, transcription, post-transcriptional modification, translation, and post-translational modification. In some embodiments, the term also includes secretion of the polypeptide from a cell.

[0091] "Host cell" and "host strain" refer to a suitable host for an expression vector comprising the DNA provided herein (e.g., a polynucleotide sequence encoding at least one neprosin polypeptide). In some embodiments, the host cell is a prokaryotic or eukaryotic cell that has been transformed or transfected with a vector constructed using recombinant DNA techniques as known in the art.

[0092] An "effective amount" refers to an amount sufficient to produce the desired result. One of ordinary skill in the art can determine what the effective amount is by using routine experiments.

[0093] "Isolated" and "purified" are used to refer to a molecule (e.g., an isolated nucleic acid, polypeptide, etc.) or other component that is separated from at least one other component with which it is naturally associated. The term "purified" does not require absolute purity, but is intended as a relative definition.

[0094] "Subject" includes mammals, such as humans, non-human primates, livestock, pets, and laboratory animals (e.g., rodents and lagomorphs). It is intended that the term include females as well as males. In some embodiments, "patient" means any subject being evaluated for a disease, treated for a disease, or experiencing a disease.

[0095] "Administration" and "administering" a composition means providing a composition of the invention to a subject (eg, to a person suffering from gluten sensitivity such as celiac disease).

[0096] "Pharmaceutically acceptable" means a material that can be administered to a subject without causing any adverse biological effect or interacting in a deleterious manner with any of the components in which it is contained and that possesses the desired biological activity.

[0097] "Excipient" refers to any pharmaceutically acceptable additive, carrier, diluent, adjuvant or other ingredient other than an active pharmaceutical ingredient. Excipients are typically included for formulation and / or administration purposes.

[0098] "Vehicle" when used with reference to a pharmaceutical composition means any of the standard pharmaceutical vehicles, buffers, and excipients such as stabilizers, preservatives and adjuvants.

[0099] When used with respect to symptoms of a disease / condition, a "therapeutically effective amount" refers to the amount and / or concentration of a compound (e.g., a recombinant neprosin polypeptide) that improves, reduces, or eliminates one or more symptoms of a disease / condition, or prevents or delays the onset of symptoms. When used with reference to a disease / condition, a "therapeutically effective amount" refers to the amount and / or concentration of a composition (e.g., a recombinant neprosin polypeptide) that improves, reduces, or eliminates the disease / condition. In some embodiments, the term is used to refer to the amount of a composition that elicits the biological (e.g., medical) response of a tissue, system, or animal subject that is sought by a researcher, physician, veterinarian, or other clinician.

[0100] As used herein, "treating" or "treatment" of a disease, disorder, or syndrome includes (i) preventing the disease, disorder, or syndrome from occurring in a subject, i.e., preventing the clinical symptoms of the disease, disorder, or syndrome from developing in an animal that may be exposed to or susceptible to the disease, disorder, or syndrome but does not yet experience or display symptoms of the disease, disorder, or syndrome; (ii) inhibiting the disease, disorder, or syndrome, i.e., preventing its development; and (iii) alleviating the disease, disorder, or syndrome, i.e., causing regression of the disease, disorder, or syndrome. Thus, the terms "treating," "treat," and "treatment" encompass both preventative (e.g., prophylactic) and palliative treatments. As known in the art, adjustments for systemic versus local delivery, age, weight, general health, sex, diet, time of administration, drug interactions, and severity of the condition may be necessary and will be determined by one of ordinary skill in the art.

[0101] As used herein, "simultaneous administration" or "co-treatment" includes administering the agents together, or administering the agents before or after each other.

[0102] "Modulate," "attenuate," or "ameliorate" means any treatment of a disease or disorder in a subject (such as a mammal), including: preventing or protecting against a disease or disorder, e.g., so that an abnormal biological response or symptom does not develop; inhibiting a disease or disorder, preventing or inhibiting the development of an abnormal biological response and / or clinical symptoms; and / or alleviating a disease or disorder, e.g., causing regression of an abnormal biological response and / or symptoms.

[0103] "Prevention" or "inhibition" refers to prophylactic treatment of a subject in need thereof. Prophylactic treatment can be achieved by providing an appropriate dose of a therapeutic agent to a subject at risk of developing a disease, thereby substantially avoiding the onset of the disease.

[0104] As used herein, "antigenic food or protein" refers to a food containing proteins that can cause an immune and / or inflammatory response in the intestine of a susceptible individual. In a preferred embodiment, the individual is a human and the food is a food intended for human consumption. Antigenic foods include, for example, but not limited to, wheat, rye, barley, peanuts, nuts, and seeds. In some embodiments, antigenic proteins from these foods include prolamins, 2S albumin, nonspecific lipid transfer protein, bifunctional α-amylase / protease inhibitor, soybean hydrophobic protein, indoline, gluten, serpin, purinin, α-amylase / protease inhibitor, globulin, and farinin. In some embodiments, the antigenic protein (or peptide) is rich in proline and / or glutamine residues. In a preferred embodiment, the antigenic protein is gluten. In some embodiments, the potentially antigenic protein is wheat protein.

[0105] "Gluten" generally refers to proteins present in wheat or related cereal species (including barley and rye), which have potential harmful effects on certain individuals. Gluten includes: gliadin such as α-gliadin, β-gliadin, γ-gliadin and ω-gliadin, which are monomeric proteins; and glutenin, which are highly heterogeneous mixtures of aggregates of high molecular weight and low molecular weight subunits held together by disulfide bonds. Many wheat gluten proteins have been characterized (see, e.g., Woychik et al., Amino Acid Composition of Proteins in Wheat Gluten, 1 Agric. Food Chem., 1961, 9 (4): 307-310). As used herein, the term gluten also includes oligopeptides that can be derived from normal human digestion of gluten from gluten-containing foods and cause abnormal immune responses. Some of these oligopeptides are resistant to normal digestive enzymes. Gluten, including the above-mentioned proteins and oligopeptides, is considered to act as an antigen of T cells (e.g., IEL) in patients with gluten intolerance (e.g., steatorrhea). The term gluten also refers to denatured gluten, such as found in baked products.

[0106] "Gluten sensitivity and related disorders" refers to any condition that stems from an intolerance or sensitivity to gluten proteins or peptides. These include, but are not limited to, steatorrhea (celiac disease), wheat allergy, gluten sensitivity, gluten-sensitive enteropathy, idiopathic gluten sensitivity, and dermatitis herpetiformis. Related disorders also include, but are not limited to, autism, attention deficit hyperactivity disorder (ADHD), rheumatoid arthritis, fibromyalgia, Crohn's disease, nutrient malabsorption, and irritable bowel syndrome (IBS).

[0107] "Manifestations of celiac disease" refers to any symptom or clinical manifestation of celiac disease. Such manifestations include, but are not limited to, intestinal inflammation, "foggy mind", depression, anxiety, ADHD-like behavior, abdominal pain, bloating, diarrhea, constipation, headache, migraine, bone or joint pain, chronic fatigue, small intestinal damage, production of tissue transglutaminase (tTG) antibodies, severe acne, vomiting, weight loss, irritability, iron deficiency anemia, arthritis, tingling and numbness in the limbs, infertility, and oral ulcers. Manifestations also include villous atrophy of the small intestinal mucosa with crypt hyperplasia, intestinal mucosal inflammation, malabsorption of nutrients, abdominal distension, and a significantly increased risk of osteoporosis and intestinal malignancies (lymphoma and epithelial cancer). Recombinant neprosin polypeptide and expressed neprosin polypeptide

[0108] Nepenthes, commonly known as monkey cups, is a carnivorous pitcher plant that grows in tropical regions. Nepenthes secretions contain a variety of different proteases. Concentrated Nepenthes fluid has high specificity for gluten peptides rich in proline and glutamine. U.S. Patent Publication Nos. 2014 / 0186330 and 2014 / 0140980, which are incorporated herein by reference in their entirety, describe the activity and specificity of concentrated Nepenthes fluid and recombinant Nepenthes enzymes. Nepenthes fluid is acidic, and the enzymes therein are generally most active at acidic pH.

[0109] The prolyl endoprotease neprosin is one of the proteases present in Nepenthes secretions. It has high proteolytic activity and is used to cleave proline-rich proteins and oligopeptides such as gluten. The amino acid sequence of the neprosin polypeptide (SEQ ID NO: 1) present in N. xventrata is Figure 1A As shown in . The neprosin polypeptide comprises a signal sequence, a pro-domain region, and a neprosin protease region. As disclosed herein, analysis of pH-activated mature, proteolytically active neprosin polypeptides by N-terminal sequencing indicated that the propolypeptide is cleaved between the proline and serine residues at amino acid positions 128 and 129 of SEQ ID NO: 1 at acidic pH, such that the primary form of the mature, proteolytically active neprosin polypeptide produced at acidic pH begins at the serine residue at amino acid position 129. Without being bound by any theory of operation, the N-glycosylation site at Figure 1A The propolypeptide form of the neprosin polypeptide (SEQ ID NO: 8) is shown in Figure 1B, the prodomain (pro-peptide) and the protease region (Neprosin 1) are shown by box arrows. N-glycosylation sites, i.e., asparagine residues (N), are present at amino acid positions 44, 121, 128, and 229 with reference to SEQ ID NO: 8, which are equivalent to amino acid positions 68, 145, 152, and 253 with reference to SEQ ID NO: 1.

[0110] Neprosin is active over a wide pH range and is particularly active at low pH (e.g., about 3 to 5), and cleaves peptides at the carboxyl (C) terminal side of proline. This cleavage appears to be highly specific. The activity and specificity of neprosin in cleaving gluten into peptides with reduced immunogenicity provides a basis for the treatment of celiac disease and other inflammatory disorders associated with the immunogenicity of gluten. Therefore, the present disclosure provides recombinant neprosin polypeptides and methods of expressing recombinant neprosin polypeptides (particularly soluble forms). In some embodiments, the recombinant neprosin polypeptide is in propolypeptide form. In some embodiments, the propolypeptide form can be cleaved, such as at an acidic pH or by protease cleavage, to form a mature polypeptide.

[0111] In some embodiments, recombinant neprosin is glycosylated. In some embodiments, the amino acid sequence of the recombinant neprosin polypeptide is manipulated to control the glycosylation of the neprosin polypeptide. In some embodiments, the propolypeptide of the recombinant neprosin polypeptide has an altered glycosylation. In some embodiments, the mature polypeptide of the recombinant neprosin polypeptide has an altered glycosylation pattern compared to the naturally occurring polypeptide. In some embodiments, the glycosylation pattern is manipulated by selecting a host cell (e.g., a mammalian cell, an insect cell, or a fungal cell) to express the recombinant neprosin polypeptide.

[0112] In some embodiments, the recombinant neprosin polypeptide lacks glycosylation at amino acid position 68, 145, 152 or 253, or the equivalent thereof, and combinations thereof, wherein the amino acid positions are relative to SEQ ID NO: 1. In some embodiments, the recombinant neprosin polypeptide lacks glycosylation at least at amino acid position 68, or the equivalent thereof. In some embodiments, the recombinant neprosin polypeptide lacks glycosylation at least at amino acid position 145, or the equivalent thereof. In some embodiments, the recombinant neprosin polypeptide lacks glycosylation at least at amino acid position 152, or the equivalent thereof. In some embodiments, the recombinant neprosin polypeptide lacks glycosylation at least at amino acid position 253, or the equivalent thereof. In some embodiments, the recombinant neprosin polypeptide is glycosylated, but lacks glycosylation at least at amino acid position 68, 145, 152 or 253, or the equivalent thereof, wherein the amino acid positions are relative to SEQ ID NO: 1.

[0113] In some embodiments, the recombinant neprosin polypeptide is non-glycosylated. In some embodiments, the pre-propeptide of recombinant neprosin is non-glycosylated. In some embodiments, the pro-polypeptide form of recombinant neprosin is non-glycosylated. In some embodiments, the mature polypeptide of recombinant neprosin is non-glycosylated.

[0114] In one aspect, the disclosure provides a recombinant neprosin polypeptide comprising an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence of a prepropolypeptide, propolypeptide, mature polypeptide or polypeptide corresponding to SEQ ID NO: 1, wherein the amino acid sequence comprises a substitution with an amino acid other than asparagine (N) at at least amino acid position 68, 145, 152 or 253, or a combination thereof and the equivalent thereof, wherein the amino acid position is relative to the reference sequence corresponding to SEQ ID NO: 1. In some embodiments, the amino acid substitution or amino acid residue at position 68, 145, 152 or 253 is independently selected from A, C, D, E, F, G, H, I, K, L, M, P, Q, R, S, T, V, W and Y. In some embodiments, the amino acid substitutions or amino acid residues at positions 68, 145, 152, and 253 are each A.

[0115] In some embodiments, the recombinant neprosin polypeptide comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence of a prepropolypeptide, propolypeptide, mature polypeptide, or polypeptide corresponding to SEQ ID NO: 1, wherein the amino acid sequence comprises a substitution with an amino acid other than asparagine (N) at least at amino acid position 68, or the equivalent thereof, wherein the amino acid position is relative to the reference sequence corresponding to SEQ ID NO: 1. In some embodiments, the amino acid substitution or amino acid residue at position 68 is selected from A, C, D, E, F, G, H, I, K, L, M, P, Q, R, S, T, V, W, and Y. In some embodiments, the amino acid substitution or amino acid residue at position 68 is A.

[0116] In some embodiments, the recombinant neprosin polypeptide comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence corresponding to a prepropolypeptide, propolypeptide, mature polypeptide, or polypeptide of SEQ ID NO: 1, wherein the amino acid sequence comprises a substitution with an amino acid other than asparagine (N) at least at amino acid position 145, or the equivalent thereof, wherein the amino acid position is relative to SEQ ID NO: 1. In some embodiments, the amino acid substitution or amino acid residue at position 145 is selected from A, C, D, E, F, G, H, I, K, L, M, P, Q, R, S, T, V, W, and Y. In some embodiments, the amino acid substitution or amino acid residue at position 145 is A.

[0117] In some embodiments, the recombinant neprosin polypeptide comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence corresponding to a prepropolypeptide, propolypeptide, mature polypeptide, or polypeptide of SEQ ID NO: 1, wherein the amino acid sequence comprises a substitution with an amino acid other than asparagine (N) at least at amino acid position 152, or the equivalent thereof, wherein the amino acid position is relative to SEQ ID NO: 1. In some embodiments, the amino acid substitution or amino acid residue at position 152 is selected from A, C, D, E, F, G, H, I, K, L, M, P, Q, R, S, T, V, W, and Y. In some embodiments, the amino acid substitution or amino acid residue at position 152 is A.

[0118] In some embodiments, the recombinant neprosin polypeptide comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence corresponding to a prepropolypeptide, propolypeptide, mature polypeptide, or polypeptide of SEQ ID NO: 1, wherein the amino acid sequence comprises a substitution with an amino acid other than asparagine (N) at at least amino acid position 253, or the equivalent thereof, wherein the amino acid position is relative to SEQ ID NO: 1. In some embodiments, the amino acid substitution or amino acid residue at position 253 is selected from A, C, D, E, F, G, H, I, K, L, M, P, Q, R, S, T, V, W, and Y. In some embodiments, the amino acid substitution or amino acid residue at position 253 is A.

[0119] In some embodiments, the recombinant neprosin polypeptide comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence corresponding to a prepropolypeptide, propolypeptide, mature polypeptide, or polypeptide of SEQ ID NO: 1, wherein the amino acid sequence comprises a substitution with an amino acid other than asparagine (N) at at least amino acid positions 68 and 145, or the equivalent thereof, wherein the amino acid positions are relative to SEQ ID NO: 1. In some embodiments, the amino acid substitution or amino acid residue at each of positions 68 and 145 is independently selected from A, C, D, E, F, G, H, I, K, L, M, P, Q, R, S, T, V, W, and Y. In some embodiments, the amino acid substitution or amino acid residue at positions 68 and 145 is A.

[0120] In some embodiments, the recombinant neprosin polypeptide comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence corresponding to a prepropolypeptide, propolypeptide, mature polypeptide, or polypeptide of SEQ ID NO: 1, wherein the amino acid sequence comprises a substitution with an amino acid other than asparagine (N) at at least amino acid positions 68 and 152, or the equivalent thereof, wherein the amino acid positions are relative to SEQ ID NO: 1. In some embodiments, the amino acid substitution or amino acid residue at each of positions 68 and 152 is independently selected from A, C, D, E, F, G, H, I, K, L, M, P, Q, R, S, T, V, W, and Y. In some embodiments, the amino acid substitution or amino acid residue at positions 68 and 152 is A.

[0121] In some embodiments, the recombinant neprosin polypeptide comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence corresponding to a prepropolypeptide, propolypeptide, mature polypeptide, or polypeptide of SEQ ID NO: 1, wherein the amino acid sequence comprises a substitution with an amino acid other than asparagine (N) at at least amino acid positions 68 and 253, or the equivalent thereof, wherein the amino acid positions are relative to SEQ ID NO: 1. In some embodiments, the amino acid substitution or amino acid residue at each of positions 68 and 253 is independently selected from A, C, D, E, F, G, H, I, K, L, M, P, Q, R, S, T, V, W, and Y. In some embodiments, the amino acid substitution or amino acid residue at positions 68 and 253 is A.

[0122] In some embodiments, the recombinant neprosin polypeptide comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence corresponding to a prepropolypeptide, propolypeptide, mature polypeptide, or polypeptide of SEQ ID NO: 1, wherein the amino acid sequence comprises a substitution with an amino acid other than asparagine (N) at at least amino acid positions 145 and 152, or the equivalent thereof, wherein the amino acid positions are relative to SEQ ID NO: 1. In some embodiments, the amino acid substitution or amino acid residue at each of positions 145 and 152 is independently selected from A, C, D, E, F, G, H, I, K, L, M, P, Q, R, S, T, V, W, and Y. In some embodiments, the amino acid substitution or amino acid residue at positions 145 and 152 is A.

[0123] In some embodiments, the recombinant neprosin polypeptide comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence corresponding to a prepropolypeptide, propolypeptide, mature polypeptide or polypeptide of SEQ ID NO: 1, wherein the amino acid sequence comprises a substitution with an amino acid other than asparagine (N) at at least amino acid positions 145 and 253, or the equivalent thereof, wherein the amino acid positions are relative to SEQ ID NO: 1. In some embodiments, the amino acid substitution or amino acid residue at each of positions 145 and 253 is independently selected from A, C, D, E, F, G, H, I, K, L, M, P, Q, R, S, T, V, W and Y. In some embodiments, the amino acid substitution or amino acid residue at positions 145 and 253 is A.

[0124] In some embodiments, the recombinant neprosin polypeptide comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence corresponding to a prepropolypeptide, propolypeptide, mature polypeptide, or polypeptide of SEQ ID NO: 1, wherein the amino acid sequence comprises a substitution with an amino acid other than asparagine (N) at at least amino acid positions 152 and 253, or the equivalent thereof, wherein the amino acid positions are relative to SEQ ID NO: 1. In some embodiments, the amino acid substitution or amino acid residue at each of positions 152 and 253 is independently selected from A, C, D, E, F, G, H, I, K, L, M, P, Q, R, S, T, V, W, and Y. In some embodiments, the amino acid substitution or amino acid residue at positions 152 and 253 is A.

[0125] In some embodiments, the recombinant neprosin polypeptide comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence corresponding to a prepropolypeptide, propolypeptide, mature polypeptide, or polypeptide of SEQ ID NO: 1, wherein the amino acid sequence comprises a substitution with an amino acid other than asparagine (N) at at least amino acid positions 68, 145, and 152, or the equivalent thereof, wherein the amino acid positions are relative to SEQ ID NO: 1. In some embodiments, the amino acid substitution or amino acid residue at each of positions 68, 145, and 152 is independently selected from A, C, D, E, F, G, H, I, K, L, M, P, Q, R, S, T, V, W, and Y. In some embodiments, the amino acid substitution or amino acid residue at positions 68, 145, and 152 is A.

[0126] In some embodiments, the recombinant neprosin polypeptide comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence corresponding to a prepropolypeptide, propolypeptide, mature polypeptide or polypeptide of SEQ ID NO: 1, wherein the amino acid sequence comprises a substitution with an amino acid other than asparagine (N) at at least amino acid positions 68, 145 and 253, or the equivalent thereof, wherein the amino acid positions are relative to SEQ ID NO: 1. In some embodiments, the amino acid substitution or amino acid residue at each of positions 68, 145 and 253 is independently selected from A, C, D, E, F, G, H, I, K, L, M, P, Q, R, S, T, V, W and Y. In some embodiments, the amino acid substitution or amino acid residue at positions 68, 145 and 253 is A.

[0127] In some embodiments, the recombinant neprosin polypeptide comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence corresponding to a prepropolypeptide, propolypeptide, mature polypeptide or polypeptide of SEQ ID NO: 1, wherein the amino acid sequence comprises a substitution with an amino acid other than asparagine (N) at at least amino acid positions 68, 152 and 253, or the equivalent thereof, wherein the amino acid positions are relative to SEQ ID NO: 1. In some embodiments, the amino acid substitution or amino acid residue at each of positions 68, 152 and 253 is independently selected from A, C, D, E, F, G, H, I, K, L, M, P, Q, R, S, T, V, W and Y. In some embodiments, the amino acid substitution or amino acid residue at positions 68, 152 and 253 is A.

[0128] In some embodiments, the recombinant neprosin polypeptide comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence corresponding to a prepropolypeptide, propolypeptide, mature polypeptide, or polypeptide of SEQ ID NO: 1, wherein the amino acid sequence comprises a substitution with an amino acid other than asparagine (N) at at least amino acid positions 145, 152, and 253, or the equivalent thereof, wherein the amino acid positions are relative to SEQ ID NO: 1. In some embodiments, the amino acid substitution or amino acid residue at each of positions 145, 152, and 253 is independently selected from A, C, D, E, F, G, H, I, K, L, M, P, Q, R, S, T, V, W, and Y. In some embodiments, the amino acid substitution or amino acid residue at positions 145, 152, and 253 is A.

[0129] In some embodiments, the recombinant neprosin polypeptide comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence corresponding to a prepropolypeptide, propolypeptide, mature polypeptide or polypeptide of SEQ ID NO: 1, wherein the amino acid sequence comprises a substitution with an amino acid other than asparagine (N) at at least amino acid positions 68, 145, 152 and 253, or the equivalent thereof, wherein the amino acid positions are relative to SEQ ID NO: 1. In some embodiments, the amino acid substitution or amino acid residue at each of positions 68, 145, 152 and 253 is independently selected from A, C, D, E, F, G, H, I, K, L, M, P, Q, R, S, T, V, W and Y. In some embodiments, the amino acid substitution or amino acid residue at positions 68, 145, 152, and 253 is A.

[0130] In some embodiments, the recombinant neprosin polypeptide comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence corresponding to a prepropolypeptide, propolypeptide, mature polypeptide or polypeptide of SEQ ID NO: 1, wherein the amino acid sequence comprises at least the substitution N68A, N145A, N152A or N253A, or the equivalent thereof, and combinations thereof, wherein the amino acid positions are relative to SEQ ID NO: 1.

[0131] In some embodiments, the recombinant neprosin polypeptide comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence corresponding to a prepropolypeptide, propolypeptide, mature polypeptide or polypeptide of SEQ ID NO: 1, wherein the amino acid sequence comprises at least the substitution N68A or the equivalent thereof.

[0132] In some embodiments, the recombinant neprosin polypeptide comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence corresponding to a prepropolypeptide, propolypeptide, mature polypeptide or polypeptide of SEQ ID NO: 1, wherein the amino acid sequence comprises at least the substitution N145A or the equivalent thereof.

[0133] In some embodiments, the recombinant neprosin polypeptide comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence corresponding to a prepropolypeptide, propolypeptide, mature polypeptide or polypeptide of SEQ ID NO: 1, wherein the amino acid sequence comprises at least the substitution N152A or the equivalent thereof.

[0134] In some embodiments, the recombinant neprosin polypeptide comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence corresponding to a prepropolypeptide, propolypeptide, mature polypeptide or polypeptide of SEQ ID NO: 1, wherein the amino acid sequence comprises at least the substitution N253A or the equivalent thereof.

[0135] In some embodiments, the recombinant neprosin polypeptide comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence corresponding to a prepropolypeptide, propolypeptide, mature polypeptide or polypeptide of SEQ ID NO: 1, wherein the amino acid sequence comprises at least the substitutions N68A and N152A or the equivalent thereof.

[0136] In some embodiments, the recombinant neprosin polypeptide comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence corresponding to a prepropolypeptide, propolypeptide, mature polypeptide or polypeptide of SEQ ID NO: 1, wherein the amino acid sequence comprises at least the substitutions N68A and N253A or the equivalent thereof.

[0137] In some embodiments, the recombinant neprosin polypeptide comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence corresponding to a prepropolypeptide, propolypeptide, mature polypeptide or polypeptide of SEQ ID NO: 1, wherein the amino acid sequence comprises at least substitutions N145A and N253A or the equivalent thereof.

[0138] In some embodiments, the recombinant neprosin polypeptide comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence corresponding to a prepropolypeptide, propolypeptide, mature polypeptide or polypeptide of SEQ ID NO: 1, wherein the amino acid sequence comprises at least substitutions N152A and N253A or the equivalent thereof.

[0139] In some embodiments, the recombinant neprosin polypeptide comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence corresponding to a prepropolypeptide, propolypeptide, mature polypeptide or polypeptide of SEQ ID NO: 1, wherein the amino acid sequence comprises at least the substitutions N68A, N152A and N253A or the equivalent thereof.

[0140] In some embodiments, the recombinant neprosin polypeptide comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence corresponding to a prepropolypeptide, propolypeptide, mature polypeptide or polypeptide of SEQ ID NO: 1, wherein the amino acid sequence comprises at least the substitutions N145A, N152A and N253A or the equivalent thereof.

[0141] In some embodiments, the recombinant neprosin polypeptide comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence corresponding to a prepropolypeptide, propolypeptide, mature polypeptide or polypeptide of SEQ ID NO: 1, wherein the amino acid sequence comprises at least the substitutions N68A, N145A, N152A and N253A or the equivalent thereof.

[0142] In some embodiments, the recombinant neprosin polypeptide comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a polypeptide sequence corresponding to residues 26 to 381 of SEQ ID NO: 44, or a pro- or mature polypeptide thereof, wherein the amino acid at position 68, 152, and / or 253 is not asparagine (N).

[0143] In some embodiments, the recombinant neprosin polypeptide comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence corresponding to a polypeptide sequence comprising residues 26 to 381 of SEQ ID NO: 46, or a pro- or mature polypeptide thereof, wherein the amino acid at position 68 is not asparagine (N).

[0144] In some embodiments, the recombinant neprosin polypeptide comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence corresponding to a polypeptide sequence comprising residues 26 to 381 of SEQ ID NO: 48, or a pro- or mature polypeptide thereof, wherein the amino acid at position 152 is not asparagine (N).

[0145] In some embodiments, the recombinant neprosin polypeptide comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence corresponding to a polypeptide sequence comprising residues 26 to 381 of SEQ ID NO: 50, or a pro- or mature polypeptide thereof, wherein the amino acid at position 253 is not asparagine (N).

[0146] In some embodiments, the recombinant neprosin polypeptide comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a polypeptide sequence comprising residues 26 to 381 of SEQ ID NO: 52, or a pro-polypeptide or mature polypeptide thereof, wherein the amino acids at positions 68 and 253 are not asparagine (N). In some embodiments, the amino acids at positions 68 and 253 are alanine (A).

[0147] In some embodiments, the recombinant neprosin polypeptide comprises an amino acid sequence comprising residues 26 to 381 of SEQ ID NO: 44, 46, 48, 50, or 52. In some embodiments, the recombinant neprosin polypeptide comprises a propolypeptide comprising an amino acid sequence comprising amino acid residues 26 to 381 of SEQ ID NO: 44, 46, 48, 50, or 52. In some embodiments, the recombinant neprosin polypeptide comprises a mature polypeptide comprising an amino acid sequence comprising amino acid residues 26 to 381 of SEQ ID NO: 44, 46, 48, 50, or 52.

[0148] In some embodiments, the recombinant neprosin polypeptide comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence corresponding to residues 105 to 356 of SEQ ID NO: 8, or to a reference sequence corresponding to SEQ ID NO: 8, wherein the amino acid sequence comprises a substitution with an amino acid other than asparagine (N) at at least amino acid position 44, 121, 128 or 229, or a combination thereof, wherein the amino acid positions are relative to SEQ ID NO: 8. In some embodiments, the amino acid substitution or amino acid residue at amino acid position 44, 121, 128 or 229, or a combination thereof, is independently selected from A, C, D, E, F, G, H, I, K, L, M, P, Q, R, S, T, V, W, and Y. In some embodiments, the amino acid substitution or amino acid residue at position 44, 121, 128 or 229, or a combination thereof, is A.

[0149] In some embodiments, the recombinant neprosin polypeptide comprises an amino acid sequence comprising residues 105 to 356 of SEQ ID NO: 8, wherein the amino acid sequence comprises a substitution with an amino acid other than asparagine (N) at least at amino acid position 121, 128, or 229, or a combination thereof, wherein the amino acid positions are relative to SEQ ID NO: 8. In some embodiments, the recombinant neprosin polypeptide comprises an amino acid sequence comprising SEQ ID NO: 8, wherein the amino acid sequence comprises a substitution with an amino acid other than asparagine (N) at least at amino acid position 44, 121, 128, or 229, or a combination thereof, wherein the amino acid positions are relative to SEQ ID NO: 8. In some embodiments, the amino acid substitution or amino acid residue at amino acid position 44, 121, 128, or 229, or a combination thereof, is independently selected from A, C, D, E, F, G, H, I, K, L, M, P, Q, R, S, T, V, W, and Y. In some embodiments, the amino acid substitution or amino acid residue at position 44, 121, 128 or 229, or a combination thereof, is A.

[0150] In some embodiments, the recombinant neprosin polypeptide comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence corresponding to residues 105 to 356 of SEQ ID NO: 8, or to a reference sequence corresponding to SEQ ID NO: 8, wherein the amino acid sequence comprises a substitution with an amino acid other than asparagine (N) at at least amino acid position 44, wherein the amino acid positions are relative to SEQ ID NO: 8. In some embodiments, the amino acid substitution or amino acid residue at amino acid position 44 is selected from A, C, D, E, F, G, H, I, K, L, M, P, Q, R, S, T, V, W, and Y. In some embodiments, the amino acid substitution or amino acid residue at position 44 is A.

[0151] In some embodiments, the recombinant neprosin polypeptide comprises an acid activated proteolytically active polypeptide derived from or comprising a subsequence of SEQ ID NO: 10. In some embodiments, the recombinant neprosin polypeptide comprises an amino acid sequence comprising residues 105 to 356 of SEQ ID NO: 10. In some embodiments, the recombinant neprosin polypeptide comprises an amino acid sequence comprising SEQ ID NO: 10. In some embodiments, the recombinant neprosin polypeptide comprising residues 105 to 356 of SEQ ID NO: 10 or comprising an amino acid sequence of SEQ ID NO: 10 is glycosylated.

[0152] In some embodiments, the recombinant neprosin polypeptide comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence corresponding to residues 105 to 356 of SEQ ID NO: 8, or to a reference sequence corresponding to SEQ ID NO: 8, wherein the amino acid sequence comprises a substitution with an amino acid other than asparagine (N) at at least amino acid position 121, wherein the amino acid position is relative to SEQ ID NO: 8. In some embodiments, the amino acid substitution or amino acid residue at position 121 is selected from A, C, D, E, F, G, H, I, K, L, M, P, Q, R, S, T, V, W, and Y. In some embodiments, the amino acid substitution or amino acid residue at position 121 is A.

[0153] In some embodiments, the recombinant neprosin polypeptide comprises an acid-activated proteolytically active polypeptide derived from a subsequence of SEQ ID NO: 12 or comprising a subsequence of SEQ ID NO: 12. In some embodiments, the recombinant neprosin polypeptide comprises an amino acid sequence comprising residues 105 to 356 of SEQ ID NO: 12. In some embodiments, the recombinant neprosin polypeptide comprises an amino acid sequence comprising SEQ ID NO: 12. In some embodiments, the recombinant neprosin polypeptide comprising residues 105 to 356 of SEQ ID NO: 12 or comprising an amino acid sequence of SEQ ID NO: 12 is glycosylated.

[0154] In some embodiments, the recombinant neprosin polypeptide comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence corresponding to residues 105 to 356 of SEQ ID NO: 8, or to a reference sequence corresponding to SEQ ID NO: 8, wherein the amino acid sequence comprises a substitution with an amino acid other than asparagine (N) at at least amino acid position 128, wherein the amino acid position is relative to SEQ ID NO: 8. In some embodiments, the amino acid substitution or amino acid residue at position 128 is selected from A, C, D, E, F, G, H, I, K, L, M, P, Q, R, S, T, V, W, and Y. In some embodiments, the amino acid substitution or amino acid residue at position 128 is A.

[0155] In some embodiments, the recombinant neprosin polypeptide comprises an acid-activated proteolytically active polypeptide derived from a subsequence of or comprising SEQ ID NO: 14. In some embodiments, the recombinant neprosin polypeptide comprises an amino acid sequence comprising residues 105 to 356 of SEQ ID NO: 14. In some embodiments, the recombinant neprosin polypeptide comprises an amino acid sequence comprising SEQ ID NO: 14. In some embodiments, the recombinant neprosin polypeptide comprising residues 105 to 356 of or comprising SEQ ID NO: 14 is glycosylated.

[0156] In some embodiments, the recombinant neprosin polypeptide comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence corresponding to residues 105 to 356 of SEQ ID NO: 8, or to a reference sequence corresponding to SEQ ID NO: 8, wherein the amino acid sequence comprises a substitution with an amino acid other than asparagine (N) at at least amino acid position 229, wherein the amino acid position is relative to SEQ ID NO: 8. In some embodiments, the amino acid substitution or amino acid residue at position 229 is selected from A, C, D, E, F, G, H, I, K, L, M, P, Q, R, S, T, V, W, and Y. In some embodiments, the amino acid substitution or amino acid residue at position 229 is A.

[0157] In some embodiments, the recombinant neprosin polypeptide comprises an acid-activated proteolytically active polypeptide derived from a subsequence of or comprising SEQ ID NO: 16. In some embodiments, the recombinant neprosin polypeptide comprises an amino acid sequence comprising residues 105 to 356 of SEQ ID NO: 16. In some embodiments, the recombinant neprosin polypeptide comprises an amino acid sequence comprising SEQ ID NO: 16. In some embodiments, the recombinant neprosin polypeptide comprising residues 105 to 356 of SEQ ID NO: 12 or an amino acid sequence comprising SEQ ID NO: 16 is glycosylated.

[0158] In some embodiments, the recombinant neprosin polypeptide comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence corresponding to residues 105 to 356 of SEQ ID NO: 8, or to a reference sequence corresponding to SEQ ID NO: 8, wherein the amino acid sequence comprises a substitution with an amino acid other than asparagine (N) at at least amino acid positions 121 and 229, wherein the amino acid positions are relative to SEQ ID NO: 8. In some embodiments, the amino acid substitution or amino acid residue at each of positions 121 and 229 is independently selected from A, C, D, E, F, G, H, I, K, L, M, P, Q, R, S, T, V, W, and Y. In some embodiments, the amino acid substitution or amino acid residue at positions 121 and 229 is A.

[0159] In some embodiments, the recombinant neprosin polypeptide comprises an acid-activated proteolytically active polypeptide derived from a subsequence of or comprising SEQ ID NO: 18. In some embodiments, the recombinant neprosin polypeptide comprises an amino acid sequence comprising residues 105 to 356 of SEQ ID NO: 18. In some embodiments, the recombinant neprosin polypeptide comprises an amino acid sequence comprising SEQ ID NO: 18. In some embodiments, the recombinant neprosin polypeptide comprising residues 105 to 356 of or comprising SEQ ID NO: 18 is glycosylated.

[0160] In some embodiments, the recombinant neprosin polypeptide comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence corresponding to residues 105 to 356 of SEQ ID NO: 8, or to a reference sequence corresponding to SEQ ID NO: 8, wherein the amino acid sequence comprises a substitution with an amino acid other than asparagine (N) at at least amino acid positions 44, 128, and 229, or the equivalent thereof, wherein the amino acid positions are relative to SEQ ID NO: 8. In some embodiments, the amino acid substitution or amino acid residue at each of positions 44, 128, and 229 is independently selected from A, C, D, E, F, G, H, I, K, L, M, P, Q, R, S, T, V, W, and Y. In some embodiments, the amino acid substitution or amino acid residue at positions 44, 128, and 229 is A.

[0161] In some embodiments, the recombinant neprosin polypeptide comprises an acid-activated proteolytically active polypeptide derived from a subsequence of SEQ ID NO: 20 or comprising a subsequence of SEQ ID NO: 20. In some embodiments, the recombinant neprosin polypeptide comprises an amino acid sequence comprising residues 105 to 356 of SEQ ID NO: 20. In some embodiments, the recombinant neprosin polypeptide comprises an amino acid sequence comprising SEQ ID NO: 20. In some embodiments, the recombinant neprosin polypeptide comprising residues 105 to 356 of SEQ ID NO: 20 or comprising an amino acid sequence of SEQ ID NO: 20 is glycosylated.

[0162] In some embodiments, the recombinant neprosin polypeptide comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence corresponding to residues 105 to 356 of SEQ ID NO: 8, or to a reference sequence corresponding to SEQ ID NO: 8, wherein the amino acid sequence comprises a substitution with an amino acid other than asparagine (N) at at least amino acid positions 44, 121, 128, and 229, wherein the amino acid positions are relative to SEQ ID NO: 8. In some embodiments, the amino acid substitution or amino acid residue at each of positions 44, 121, 128, and 229 is independently selected from A, C, D, E, F, G, H, I, K, L, M, P, Q, R, S, T, V, W, and Y. In some embodiments, the amino acid substitution or amino acid residue at positions 44, 121, 128, and 229 is A.

[0163] In some embodiments, the recombinant neprosin polypeptide comprises an acid-activated proteolytically active polypeptide derived from or comprising a subsequence of SEQ ID NO: 22. In some embodiments, the recombinant neprosin polypeptide comprises an amino acid sequence comprising residues 105 to 356 of SEQ ID NO: 22. In some embodiments, the recombinant neprosin polypeptide comprises an amino acid sequence comprising SEQ ID NO: 22. In some embodiments, the recombinant neprosin polypeptide comprising residues 105 to 356 of SEQ ID NO: 22 or comprising an amino acid sequence of SEQ ID NO: 22 is not glycosylated.

[0164] In some embodiments, the recombinant neprosin polypeptide comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence corresponding to residues 105 to 356 of SEQ ID NO: 8, or to a reference sequence corresponding to SEQ ID NO: 8, wherein the amino acid sequence comprises a substitution with an amino acid other than asparagine (N) at at least amino acid positions 44 and 229, wherein the amino acid positions are relative to SEQ ID NO: 8. In some embodiments, the amino acid substitution or amino acid residue at each of positions 44 and 229 is independently selected from A, C, D, E, F, G, H, I, K, L, M, P, Q, R, S, T, V, W, and Y. In some embodiments, the amino acid substitution or amino acid residue at positions 44 and 229 is A.

[0165] In some embodiments, the recombinant neprosin polypeptide comprises an acid-activated proteolytically active polypeptide derived from a subsequence of SEQ ID NO: 32 or comprising a subsequence of SEQ ID NO: 32. In some embodiments, the recombinant neprosin polypeptide comprises an amino acid sequence comprising residues 105 to 356 of SEQ ID NO: 32. In some embodiments, the recombinant neprosin polypeptide comprises an amino acid sequence comprising SEQ ID NO: 32. In some embodiments, the recombinant neprosin polypeptide comprising residues 105 to 356 of SEQ ID NO: 32 or comprising an amino acid sequence of SEQ ID NO: 32 is glycosylated.

[0166] In some embodiments, the recombinant neprosin polypeptide comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence corresponding to residues 105 to 356 of SEQ ID NO: 8, or to a reference sequence corresponding to SEQ ID NO: 8.

[0167] In some embodiments, the recombinant neprosin polypeptide comprises an acid-activated proteolytically active polypeptide derived from a subsequence of SEQ ID NO: 8 or comprising a subsequence of SEQ ID NO: 8. In some embodiments, the recombinant neprosin polypeptide comprises an amino acid sequence comprising residues 105 to 356 of SEQ ID NO: 8. In some embodiments, the recombinant neprosin polypeptide comprises an amino acid sequence comprising SEQ ID NO: 8. In some embodiments, the recombinant neprosin polypeptide comprising residues 105 to 356 of SEQ ID NO: 8 or comprising an amino acid sequence of SEQ ID NO: 8 is glycosylated.

[0168] In some of the foregoing embodiments, the recombinant neprosin polypeptide is glycosylated. In some embodiments, the recombinant neprosin polypeptide is not glycosylated at least at amino acid position 44, 121, 128, or 229, wherein the amino acid positions are relative to SEQ ID NO: 8. In some embodiments, the recombinant neprosin polypeptide is glycosylated, but is not glycosylated at at least amino acid position 44, 121, 128, or 229, wherein the amino acid positions are relative to SEQ ID NO: 8.

[0169] In some embodiments, the recombinant neprosin polypeptide comprises an amino acid sequence comprising residues 96 to 451 of SEQ ID NO: 58, 60, 62, 64, 66, 68, or 70. In some embodiments, the recombinant neprosin polypeptide comprises a propolypeptide comprising an amino acid sequence comprising amino acid residues 96 to 451 of SEQ ID NO: 58, 60, 62, 64, 66, 68, or 70. In some embodiments, the recombinant neprosin polypeptide comprises a mature polypeptide comprising an amino acid sequence comprising amino acid residues 96 to 451 of SEQ ID NO: 58, 60, 62, 64, 66, 68, or 70.

[0170] In some embodiments, the recombinant neprosin polypeptide further comprises a signal sequence or a signal peptide. In some embodiments, the signal sequence or the signal peptide is functional in a host cell used or to be used for expressing the recombinant neprosin polypeptide. In some embodiments, the signal sequence or the signal peptide is fused to a propolypeptide form of the recombinant neprosin, for example, to form a pre-propolypeptide. In some embodiments, the signal sequence or the signal peptide is fused to a polypeptide comprising a mature active form of the recombinant neprosin. In some embodiments, the signal sequence may be a naturally occurring signal sequence or a synthetic signal sequence, including a hybrid signal sequence.

[0171] In some embodiments, the signal sequence or signal peptide is a mammalian or insect cell signal sequence or signal peptide, or a signal sequence or signal peptide that is functional in a mammalian cell or an insect cell. Exemplary mammalian or insect signal sequences or signal peptides include, human OSM, e.g., MGVLLTQRTLLSLVLALLFPSMASM (SEQ ID NO: 78); VSV-G, e.g., MKCLLYLAFLFIGVNC (SEQ ID NO: 79), mouse Ig kappa, e.g., METDTLLLWVLLLWVPGSTGD (SEQ ID NO: 80), mouse Ig heavy chain, e.g., MGWSCIILFLVATATGVHS (SEQ ID NO: 81), BM40, e.g., MRAWIFFLLCLAGRALA (SEQ ID NO: 82); Secreton, e.g., MWWRLWWLLLLLLLLWPMVWA (SEQ ID NO: 83); human IgKVIII, e.g., MDMRVPAQLLGLLLLWLRGARC (SEQ ID NO: 84); CD33, e.g., MPLLLLLPLLWAGALA (SEQ ID NO: 85). 85); tPA, e.g., MDAMKRGLCCVLLLCGAVFVSPS (SEQ ID NO: 86); human chymotrypsinogen, e.g., MAFLWLLSCWALLGTTFG (SEQ ID NO: 87); human trypsinogen-2, e.g., MNLLLILTFVAAAVA (SEQ ID NO: 88); human IL-2, e.g., MYRMQLLSCIALSLALVTNS (SEQ ID NO: 89); Gaussia luc, e.g., MGVKVLFALICIAVAEA (SEQ ID NO: 90); human serum albumin (HSA), e.g., MKWVTFISLLFSSAYS (SEQ ID NO: 91); influenza hemagglutinin, e.g., MKTIIALSYIFCLVLG (SEQ ID NO: 92); human insulin, e.g., MALWMRLLPLLALLALWGPDPAAA (SEQ ID NO: 93); silk fibroin LC, e.g., MKPIFLVLLVVTSAYA (SEQ ID NO: 94); and the honey bee melittin signal peptide of gp64 or gp67, e.g., MLLVNQSHQGFNKEHTSKMVSAIVLYVLLAAAAHSAFA (SEQ ID NO: 95), among others.In some embodiments, the mammalian signal sequence or signal peptide comprises the sequence MEWSWVFLFFLSVTTGVHS (SEQ ID NO: 96).

[0172] In some embodiments, the signal sequence or signal peptide is a fungal (e.g., yeast) signal sequence or signal peptide or a signal sequence or signal peptide that functions in a fungal cell. Exemplary fungal signal sequences or signal peptides include, Pichia pastoris Ost1, for example, MRFPSIFTAVLFAASSALAAPVNTTTEDETAQIPAEAVIGYLDLEGDFDVAVLPFSNSTNNGLLFINTTIASIAAKEEGVSLDKREAEA (SEQ ID NO: 97); Pichia pastoris Pst1, MRFPSIFTAVLFAASSALAAPANTTTFDETAQIPAFAVIDYSDLFGDFDAAALPLSNSTNNGISSTNTTIASIAAKEFGVLEKRFAFA (SEQ ID NO: 98); Saccharomyces cerevisiae (S.cerevisiae) α-mating factor, e.g., MRFPSIFTAVLFAASSALA (SEQ ID NO: 99); Saccharomyces cerevisiae invertase, e.g., MLLQAFLFLLAGF (SEQ ID NO: 100); Komagataella pastoris α-factor, e.g., MKPLILSAILITLCITSIAVSAPVEGIFADLHDSSHPYITDDVGVRMSAIKEENPDRHFVGDEIPDEAVIQSFKSKRREILFLLDDIGSAVEFIGSNLAQIEANHLSERVQFSWTHIRKNQPFGKREAEA (SEQ ID NO: 101); Saccharomyces cerevisiae CYP, e.g., MKAFTSLLCGLGLSTTLAKA (SEQ ID NO: 102); Pichia pastoris PH08, e.g., MDSEPLLPNPNDSRKPANWRRIIKYISLTLAWIGIFSYVYIYHGTA (SEQ ID NO: 103); Saccharomyces cerevisiae PEP4, e.g., MFSLKALLPLALLLVSANQVAA (SEQ ID NO: 104); Saccharomyces cerevisiae SUC2, e.g., MLLQAFLFLLAGFAAKISA (SEQ ID NO: 105); Pichia pastoris KAR2, e.g., MLSLKPSWLTLAALMYAMLLVVVPFAKPVRA (SEQ ID NO: 106); Pichia pastoris DSE4, e.g., MSFSSNVPQLFLLLVLLTNIVSG (SEQ ID NO: 107); Pichia pastoris EXG1, e.g., MNLYLITLLFASLCSA (SEQ ID NO: 108); and Pichia pastoris SCW10, e.g., MRFSNFLTVSALLTGALG (SEQ ID NO: 109), among others. In some embodiments, the signal sequence or signal peptide comprises a yeast α-mating factor pre-prosequence comprising residues 1-89 of SEQ ID NO: 54. .

[0173] Other signal sequences and signal peptides are disclosed in WO23007468, WO22192675, WO22171827, WO22090555; US2021292776, US11198881; US11306127; US8377676; US2010021967; US9273104; and Wu et al., ACS Synth. Biol., 2020, 9(8):2154-2161, among others; all references are incorporated herein by reference.

[0174] In some embodiments, the existing signal sequence or signal peptide can be replaced with another signal sequence or signal peptide, i.e., a heterologous signal sequence or signal peptide. For example, but not limited to, the signal sequence of SEQ ID NO: 1 can be replaced by an alternative sequence, such as a signal sequence for expression in mammalian or insect cells, or a signal sequence for expression in fungal cells (e.g., Pichia or Saccharomyces). In some embodiments, the signal sequence is operably fused to the propolypeptide or, in some embodiments, mature polypeptide of the recombinant neprosin described herein to promote expression and / or processing to prepare a recombinant neprosin polypeptide. Exemplary recombinant neprosin polypeptides with heterologous signal sequences or signal peptides are SEQ ID NO: 38 and SEQ ID NO: 54.

[0175] In some embodiments, the recombinant neprosin polypeptide is a fusion protein. In some embodiments, the recombinant neprosin polypeptide described herein can be fused with a variety of polypeptide sequences, such as, for example, but not limited to, polypeptide tags that can be used for detection and / or purification. In some embodiments, the fusion protein of the recombinant neprosin polypeptide comprises a glycine-histidine tag or a histidine tag (His tag). In some embodiments, the fusion protein of the recombinant neprosin polypeptide comprises an epitope tag, such as c-myc, FLAG, V5 or hemagglutinin (HA). In some embodiments, the fusion protein of the recombinant neprosin polypeptide comprises a GST, SUMO, Strep, MBP or GFP tag. In some embodiments, fused to the amino (N-) terminus of the recombinant neprosin polypeptide. In some embodiments, fused to the carboxyl (C-) terminus of the recombinant neprosin polypeptide. In some embodiments, a fusion polypeptide is inserted after the signal sequence and before the neprosin polypeptide to allow expression and secretion of a polypeptide comprising a fusion polypeptide (e.g., a polypeptide tag) and a recombinant neprosin polypeptide.

[0176] In some embodiments, the recombinant neprosin polypeptide comprises a signal sequence or a signal peptide and / or a polypeptide tag. In some embodiments, the recombinant neprosin polypeptide comprises SEQ ID NO: 36, 40, 42, 44, 46, 48, 50 or 52. In some embodiments, the recombinant neprosin polypeptide comprises SEQ ID NO: 56, 58, 60, 62, 64, 66, 68 or 70.

[0177] In some embodiments, the recombinant neprosin polypeptide is a neprosin from a species of the genus Nepenthes, including N. × ventrata, N. ampullaria, N. rafflesiana, and N. alata.

[0178] In some embodiments, the recombinant neprosin polypeptide comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a sequence corresponding to a prepropolypeptide, propolypeptide, mature polypeptide or polypeptide of SEQ ID NO: 1, 2, 3, 4, 5 or 6.

[0179] In some embodiments, the recombinant neprosin polypeptide comprises a pre-propeptide, pro-polypeptide, mature polypeptide, or polypeptide sequence corresponding to SEQ ID NO: 1, 2, 3, 4, 5, or 6. In some embodiments, the recombinant neprosin polypeptide comprises a pro-polypeptide (e.g., zymogen) of the sequence of SEQ ID NO: 1, 2, 3, 4, 5, or 6. In some embodiments, the recombinant neprosin polypeptide comprises a mature polypeptide of the sequence of SEQ ID NO: 1, 2, 3, 4, 5, or 6. In some embodiments, the recombinant neprosin polypeptide comprises an acid-activated proteolytically active polypeptide derived from a subsequence of a sequence corresponding to SEQ ID NO: 1, 2, 3, 4, 5, or 6 or containing a subsequence of a sequence corresponding to SEQ ID NO: 1, 2, 3, 4, 5, or 6.

[0180] In some embodiments, the recombinant neprosin polypeptide comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the sequence corresponding to the prepropolypeptide, propolypeptide, mature polypeptide or polypeptide of SEQ ID NO: 1.

[0181] In some embodiments, the recombinant neprosin polypeptide comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the sequence of the propolypeptide corresponding to SEQ ID NO: 1.

[0182] In some embodiments, the recombinant neprosin polypeptide comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the sequence corresponding to the mature polypeptide of SEQ ID NO: 1.

[0183] In some embodiments, the recombinant neprosin polypeptide comprises an amino acid sequence of a prepropolypeptide comprising SEQ ID NO: 1. In some embodiments, the recombinant neprosin polypeptide comprises an amino acid sequence of a propolypeptide comprising SEQ ID NO: 1. In some embodiments, the recombinant neprosin polypeptide comprises an amino acid sequence of a mature polypeptide comprising SEQ ID NO: 1. In some embodiments, the recombinant neprosin polypeptide comprises an acid-activated proteolytically active polypeptide derived from a subsequence of a sequence corresponding to SEQ ID NO: 1 or comprising a subsequence of a sequence corresponding to SEQ ID NO: 1. In some embodiments, the mature neprosin polypeptide of SEQ ID NO: 1 has a molecular weight of about 25.5 kDa to about 29 kDa, particularly as determined by SDS-PAGE.

[0184] In some embodiments, the recombinant neprosin polypeptide comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the sequence corresponding to the prepropolypeptide, propolypeptide, mature polypeptide or polypeptide of SEQ ID NO: 2.

[0185] In some embodiments, the recombinant neprosin polypeptide comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the sequence of the propolypeptide corresponding to SEQ ID NO: 2.

[0186] In some embodiments, the recombinant neprosin polypeptide comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the sequence corresponding to the mature polypeptide of SEQ ID NO: 2.

[0187] In some embodiments, the recombinant neprosin polypeptide comprises an amino acid sequence of a prepropolypeptide comprising SEQ ID NO: 2. In some embodiments, the recombinant neprosin polypeptide comprises an amino acid sequence of a propolypeptide comprising SEQ ID NO: 2. In some embodiments, the recombinant neprosin polypeptide comprises an amino acid sequence of a mature polypeptide comprising SEQ ID NO: 2. In some embodiments, the recombinant neprosin polypeptide comprises an acid-activated proteolytically active polypeptide derived from a subsequence of a sequence corresponding to SEQ ID NO: 2 or comprising a subsequence of a sequence corresponding to SEQ ID NO: 2.

[0188] In some embodiments, the recombinant neprosin polypeptide comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the sequence corresponding to the prepropolypeptide, propolypeptide, mature polypeptide or polypeptide of SEQ ID NO: 3.

[0189] In some embodiments, the recombinant neprosin polypeptide comprises an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the sequence of the propolypeptide corresponding to SEQ ID NO: 3.

[0190] In some embodiments, the recombinant neprosin polypeptide comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the sequence corresponding to the mature polypeptide of SEQ ID NO: 3.

[0191] In some embodiments, the recombinant neprosin polypeptide comprises an amino acid sequence of a prepropolypeptide comprising SEQ ID NO: 3. In some embodiments, the recombinant neprosin polypeptide comprises an amino acid sequence of a propolypeptide comprising SEQ ID NO: 3. In some embodiments, the recombinant neprosin polypeptide comprises an amino acid sequence of a mature polypeptide comprising SEQ ID NO: 3. In some embodiments, the recombinant neprosin polypeptide comprises an acid-activated proteolytically active polypeptide derived from a subsequence of a sequence corresponding to SEQ ID NO: 3 or comprising a subsequence of a sequence corresponding to SEQ ID NO: 3.

[0192] In some embodiments, the recombinant neprosin polypeptide comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the sequence corresponding to the prepropolypeptide, propolypeptide, mature polypeptide or polypeptide of SEQ ID NO: 4.

[0193] In some embodiments, the recombinant neprosin polypeptide comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the sequence of the propolypeptide corresponding to SEQ ID NO: 4.

[0194] In some embodiments, the recombinant neprosin polypeptide comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the sequence corresponding to the mature polypeptide of SEQ ID NO: 4.

[0195] In some embodiments, the recombinant neprosin polypeptide comprises an amino acid sequence of a prepropolypeptide comprising SEQ ID NO: 4. In some embodiments, the recombinant neprosin polypeptide comprises an amino acid sequence of a propolypeptide comprising SEQ ID NO: 4. In some embodiments, the recombinant neprosin polypeptide comprises an amino acid sequence of a mature polypeptide comprising SEQ ID NO: 4. In some embodiments, the recombinant neprosin polypeptide comprises an acid-activated proteolytically active polypeptide derived from a subsequence of a sequence corresponding to SEQ ID NO: 4 or comprising a subsequence of a sequence corresponding to SEQ ID NO: 4.

[0196] In some embodiments, the recombinant neprosin polypeptide comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the sequence corresponding to the prepropolypeptide, propolypeptide, mature polypeptide or polypeptide of SEQ ID NO: 5.

[0197] In some embodiments, the recombinant neprosin polypeptide comprises an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the sequence of the propolypeptide corresponding to SEQ ID NO: 5.

[0198] In some embodiments, the recombinant neprosin polypeptide comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the sequence corresponding to the mature polypeptide of SEQ ID NO: 5.

[0199] In some embodiments, the recombinant neprosin polypeptide comprises an amino acid sequence of a prepropolypeptide comprising SEQ ID NO: 5. In some embodiments, the recombinant neprosin polypeptide comprises an amino acid sequence of a propolypeptide comprising SEQ ID NO: 5. In some embodiments, the recombinant neprosin polypeptide comprises an amino acid sequence of a mature polypeptide comprising SEQ ID NO: 5. In some embodiments, the recombinant neprosin polypeptide comprises an acid-activated proteolytically active polypeptide derived from a subsequence of a sequence corresponding to SEQ ID NO: 5 or comprising a subsequence of a sequence corresponding to SEQ ID NO: 5.

[0200] In some embodiments, the recombinant neprosin polypeptide comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the sequence corresponding to the prepropolypeptide, propolypeptide, mature polypeptide or polypeptide of SEQ ID NO: 6.

[0201] In some embodiments, the recombinant neprosin polypeptide comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the sequence of the propolypeptide corresponding to SEQ ID NO: 6.

[0202] In some embodiments, the recombinant neprosin polypeptide comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the sequence corresponding to the mature polypeptide of SEQ ID NO: 6.

[0203] In some embodiments, the recombinant neprosin polypeptide comprises an amino acid sequence of a prepropolypeptide comprising SEQ ID NO: 6. In some embodiments, the recombinant neprosin polypeptide comprises an amino acid sequence of a propolypeptide comprising SEQ ID NO: 6. In some embodiments, the recombinant neprosin polypeptide comprises an amino acid sequence of a mature polypeptide comprising SEQ ID NO: 6. In some embodiments, the recombinant neprosin polypeptide comprises an acid-activated proteolytically active polypeptide derived from a subsequence of a sequence corresponding to SEQ ID NO: 6 or comprising a subsequence of a sequence corresponding to SEQ ID NO: 6. Recombinant polynucleotides, expression vectors and host cells

[0204] In another aspect, the present disclosure provides recombinant polynucleotides encoding the recombinant neprosin polypeptides described herein, and expression vectors for expressing the recombinant neprosin polypeptides in suitable host cells.

[0205] As will be apparent to the skilled artisan, the availability of protein sequences and knowledge of the codons corresponding to the various amino acids provides a description of all polynucleotides capable of encoding the subject polypeptides. The degeneracy of the genetic code, in which the same amino acid is encoded by alternative or synonymous codons, allows for the preparation of a very large number of nucleic acids, all of which encode the recombinant neprosin polypeptides of the present disclosure. Thus, the present disclosure provides methods and compositions for producing each and every possible variant of a recombinant neprosin polynucleotide encoding a recombinant neprosin polypeptide described herein by selecting a combination based on possible codon usage, and all such variants are considered to be specifically disclosed for any polypeptide described herein, including the amino acid sequence of a recombinant neprosin polypeptide described herein.

[0206] In some embodiments, codons are preferably optimized for codon utilization of the host cell selected for protein production. In some embodiments, the polynucleotides encoding the recombinant neprosin polypeptide preferably use codons used in mammalian cells, insect cells, or fungal cells. In some embodiments, the codon-optimized polynucleotides encoding the recombinant neprosin polypeptides described herein contain preferred codons at about 40%, 50%, 60%, 70%, 80%, 90%, or more than 90% of the codon positions in the coding region.

[0207] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant neprosin polypeptide comprising an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence corresponding to a prepropolypeptide, propolypeptide, mature polypeptide or polypeptide of SEQ ID NO: 1, wherein the amino acid sequence comprises a substitution with an amino acid other than asparagine (N) at at least amino acid position 68, 145, 152 or 253, or a combination thereof, and the equivalent thereof, wherein the amino acid position is relative to the reference sequence corresponding to SEQ ID NO: 1. In some embodiments, the amino acid substitution or amino acid residue at position 68, 145, 152 or 253 is independently selected from A, C, D, E, F, G, H, I, K, L, M, P, Q, R, S, T, V, W and Y. In some embodiments, the amino acid substitution or amino acid residue at position 68, 145, 152, or 253 is each A.

[0208] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant neprosin polypeptide comprising an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence corresponding to a pre-propeptide, a pro-polypeptide, a mature polypeptide, or a polypeptide of SEQ ID NO: 1, wherein the amino acid sequence comprises a substitution with an amino acid other than asparagine (N) at least at amino acid position 68, or the equivalent thereof, wherein the amino acid position is relative to the reference sequence corresponding to SEQ ID NO: 1. In some embodiments, the amino acid substitution or amino acid residue at position 68 is selected from A, C, D, E, F, G, H, I, K, L, M, P, Q, R, S, T, V, W, and Y. In some embodiments, the amino acid substitution or amino acid residue at position 68 is A.

[0209] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant neprosin polypeptide comprising an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence corresponding to a prepropolypeptide, propolypeptide, mature polypeptide, or polypeptide of SEQ ID NO: 1, wherein the amino acid sequence comprises a substitution with an amino acid other than asparagine (N) at least at amino acid position 145, or the equivalent thereof, wherein the amino acid position is relative to SEQ ID NO: 1. In some embodiments, the amino acid substitution or amino acid residue at position 145 is selected from A, C, D, E, F, G, H, I, K, L, M, P, Q, R, S, T, V, W, and Y. In some embodiments, the amino acid substitution or amino acid residue at position 145 is A.

[0210] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant neprosin polypeptide comprising an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence corresponding to a pre-propolypeptide, a pro-polypeptide, a mature polypeptide, or a polypeptide of SEQ ID NO: 1, wherein the amino acid sequence comprises a substitution with an amino acid other than asparagine (N) at amino acid position 152, or the equivalent thereof, wherein the amino acid position is relative to SEQ ID NO: 1. In some embodiments, the amino acid substitution or amino acid residue at position 152 is selected from A, C, D, E, F, G, H, I, K, L, M, P, Q, R, S, T, V, W, and Y. In some embodiments, the amino acid substitution or amino acid residue at position 152 is A.

[0211] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant neprosin polypeptide comprising an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence corresponding to a prepropolypeptide, propolypeptide, mature polypeptide, or polypeptide of SEQ ID NO: 1, wherein the amino acid sequence comprises a substitution with an amino acid other than asparagine (N) at least at amino acid position 253, or the equivalent thereof, wherein the amino acid position is relative to SEQ ID NO: 1. In some embodiments, the amino acid substitution or amino acid residue at position 253 is selected from A, C, D, E, F, G, H, I, K, L, M, P, Q, R, S, T, V, W, and Y. In some embodiments, the amino acid substitution or amino acid residue at position 253 is A.

[0212] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant neprosin polypeptide comprising an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence corresponding to a prepropolypeptide, propolypeptide, mature polypeptide or polypeptide of SEQ ID NO: 1, wherein the amino acid sequence comprises a substitution with an amino acid other than asparagine (N) at at least amino acid positions 68 and 145, or the equivalent thereof, wherein the amino acid positions are relative to SEQ ID NO: 1. In some embodiments, the amino acid substitution or amino acid residue at each of positions 68 and 145 is independently selected from A, C, D, E, F, G, H, I, K, L, M, P, Q, R, S, T, V, W and Y. In some embodiments, the amino acid substitution or amino acid residue at positions 68 and 145 is A.

[0213] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant neprosin polypeptide comprising an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence corresponding to a prepropolypeptide, propolypeptide, mature polypeptide or polypeptide of SEQ ID NO: 1, wherein the amino acid sequence comprises a substitution with an amino acid other than asparagine (N) at at least amino acid positions 68 and 152, or the equivalent thereof, wherein the amino acid positions are relative to SEQ ID NO: 1. In some embodiments, the amino acid substitution or amino acid residue at each of positions 68 and 152 is independently selected from A, C, D, E, F, G, H, I, K, L, M, P, Q, R, S, T, V, W and Y. In some embodiments, the amino acid substitution or amino acid residue at positions 68 and 152 is A.

[0214] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant neprosin polypeptide comprising an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence corresponding to a prepropolypeptide, propolypeptide, mature polypeptide or polypeptide of SEQ ID NO: 1, wherein the amino acid sequence comprises a substitution with an amino acid other than asparagine (N) at at least amino acid positions 68 and 253, or the equivalent thereof, wherein the amino acid positions are relative to SEQ ID NO: 1. In some embodiments, the amino acid substitution or amino acid residue at each of positions 68 and 253 is independently selected from A, C, D, E, F, G, H, I, K, L, M, P, Q, R, S, T, V, W and Y. In some embodiments, the amino acid substitution or amino acid residue at positions 68 and 253 is A.

[0215] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant neprosin polypeptide comprising an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence corresponding to a prepropolypeptide, propolypeptide, mature polypeptide or polypeptide of SEQ ID NO: 1, wherein the amino acid sequence comprises a substitution with an amino acid other than asparagine (N) at at least amino acid positions 145 and 152, or the equivalent thereof, wherein the amino acid positions are relative to SEQ ID NO: 1. In some embodiments, the amino acid substitution or amino acid residue at each of positions 145 and 152 is independently selected from A, C, D, E, F, G, H, I, K, L, M, P, Q, R, S, T, V, W, and Y. In some embodiments, the amino acid substitution or amino acid residue at positions 145 and 152 is A.

[0216] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant neprosin polypeptide comprising an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence corresponding to a prepropolypeptide, propolypeptide, mature polypeptide or polypeptide of SEQ ID NO: 1, wherein the amino acid sequence comprises a substitution with an amino acid other than asparagine (N) at at least the amino acids at positions 145 and 253, or the equivalent thereof, wherein the amino acid positions are relative to SEQ ID NO: 1. In some embodiments, the amino acid substitution or amino acid residue at each of positions 145 and 253 is independently selected from A, C, D, E, F, G, H, I, K, L, M, P, Q, R, S, T, V, W and Y. In some embodiments, the amino acid substitution or amino acid residue at positions 145 and 253 is A.

[0217] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant neprosin polypeptide comprising an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence corresponding to a prepropolypeptide, propolypeptide, mature polypeptide or polypeptide of SEQ ID NO: 1, wherein the amino acid sequence comprises a substitution with an amino acid other than asparagine (N) at at least amino acid positions 152 and 253, or the equivalent thereof, wherein the amino acid positions are relative to SEQ ID NO: 1. In some embodiments, the amino acid substitution or amino acid residue at each of positions 152 and 253 is independently selected from A, C, D, E, F, G, H, I, K, L, M, P, Q, R, S, T, V, W, and Y. In some embodiments, the amino acid substitution or amino acid residue at positions 152 and 253 is A.

[0218] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant neprosin polypeptide comprising an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence corresponding to a prepropolypeptide, propolypeptide, mature polypeptide or polypeptide of SEQ ID NO: 1, wherein the amino acid sequence comprises a substitution with an amino acid other than asparagine (N) at at least amino acid positions 68, 145 and 152, or the equivalent thereof, wherein the amino acid positions are relative to SEQ ID NO: 1. In some embodiments, the amino acid substitution or amino acid residue at each of positions 68, 145 and 152 is independently selected from A, C, D, E, F, G, H, I, K, L, M, P, Q, R, S, T, V, W and Y. In some embodiments, the amino acid substitution or amino acid residue at positions 68, 145, and 152 is A.

[0219] In some embodiments, the recombinant neprosin polypeptide comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence corresponding to a prepropolypeptide, propolypeptide, mature polypeptide or polypeptide of SEQ ID NO: 1, wherein the amino acid sequence comprises a substitution with an amino acid other than asparagine (N) at at least amino acid positions 68, 145 and 253, or the equivalent thereof, wherein the amino acid positions are relative to SEQ ID NO: 1. In some embodiments, the amino acid substitution or amino acid residue at each of positions 68, 145 and 253 is independently selected from A, C, D, E, F, G, H, I, K, L, M, P, Q, R, S, T, V, W and Y. In some embodiments, the amino acid substitution or amino acid residue at positions 68, 145 and 253 is A.

[0220] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant neprosin polypeptide comprising an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence corresponding to a prepropolypeptide, propolypeptide, mature polypeptide or polypeptide of SEQ ID NO: 1, wherein the amino acid sequence comprises a substitution with an amino acid other than asparagine (N) at at least amino acid positions 68, 152 and 253, or the equivalent thereof, wherein the amino acid positions are relative to SEQ ID NO: 1. In some embodiments, the amino acid substitution or amino acid residue at each of positions 68, 152 and 253 is independently selected from A, C, D, E, F, G, H, I, K, L, M, P, Q, R, S, T, V, W and Y. In some embodiments, the amino acid substitution or amino acid residue at positions 68, 152, and 253 is A.

[0221] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant neprosin polypeptide comprising an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence corresponding to a prepropolypeptide, propolypeptide, mature polypeptide or polypeptide of SEQ ID NO: 1, wherein the amino acid sequence comprises a substitution with an amino acid other than asparagine (N) at at least amino acid positions 145, 152 and 253, or the equivalent thereof, wherein the amino acid positions are relative to SEQ ID NO: 1. In some embodiments, the amino acid substitution or amino acid residue at each of positions 145, 152 and 253 is independently selected from A, C, D, E, F, G, H, I, K, L, M, P, Q, R, S, T, V, W and Y. In some embodiments, the amino acid substitution or amino acid residue at positions 145, 152, and 253 is A.

[0222] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant neprosin polypeptide comprising an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence corresponding to a prepropolypeptide, propolypeptide, mature polypeptide or polypeptide of SEQ ID NO: 1, wherein the amino acid sequence comprises a substitution with an amino acid other than asparagine (N) at at least amino acid positions 68, 145, 152 and 253, or the equivalent thereof, wherein the amino acid positions are relative to SEQ ID NO: 1. In some embodiments, the amino acid substitution or amino acid residue at each of positions 68, 145, 152 and 253 is independently selected from A, C, D, E, F, G, H, I, K, L, M, P, Q, R, S, T, V, W and Y. In some embodiments, the amino acid substitution or amino acid residue at positions 68, 145, 152, and 253 is A.

[0223] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant neprosin polypeptide comprising an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence corresponding to a prepropolypeptide, propolypeptide, mature polypeptide or polypeptide of SEQ ID NO: 1, wherein the amino acid sequence comprises at least the substitution N68A, N145A, N152A or N253A or the equivalent positions and combinations thereof, wherein the amino acid positions are relative to SEQ ID NO: 1.

[0224] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant neprosin polypeptide comprising an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence corresponding to a prepropolypeptide, propolypeptide, mature polypeptide or polypeptide of SEQ ID NO: 1, wherein the amino acid sequence comprises at least the substitution N68A or the equivalent thereof.

[0225] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant neprosin polypeptide comprising an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence corresponding to a prepropolypeptide, propolypeptide, mature polypeptide or polypeptide of SEQ ID NO: 1, wherein the amino acid sequence comprises at least the substitution N145A or the equivalent thereof.

[0226] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant neprosin polypeptide comprising an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence corresponding to a prepropolypeptide, propolypeptide, mature polypeptide or polypeptide of SEQ ID NO: 1, wherein the amino acid sequence comprises at least the substitution N152A or the equivalent thereof.

[0227] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant neprosin polypeptide comprising an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence corresponding to a prepropolypeptide, propolypeptide, mature polypeptide or polypeptide of SEQ ID NO: 1, wherein the amino acid sequence comprises at least the substitution N253A or the equivalent thereof.

[0228] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant neprosin polypeptide comprising an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence corresponding to a prepropolypeptide, propolypeptide, mature polypeptide or polypeptide of SEQ ID NO: 1, wherein the amino acid sequence comprises at least substitutions N68A and N152A or the equivalent thereof.

[0229] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant neprosin polypeptide comprising an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence corresponding to a prepropolypeptide, propolypeptide, mature polypeptide or polypeptide of SEQ ID NO: 1, wherein the amino acid sequence comprises at least the substitutions N68A and N253A or the equivalent thereof.

[0230] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant neprosin polypeptide comprising an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence corresponding to a prepropolypeptide, propolypeptide, mature polypeptide or polypeptide of SEQ ID NO: 1, wherein the amino acid sequence comprises at least substitutions N145A and N253A or the equivalent thereof.

[0231] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant neprosin polypeptide comprising an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence corresponding to a prepropolypeptide, propolypeptide, mature polypeptide or polypeptide of SEQ ID NO: 1, wherein the amino acid sequence comprises at least substitutions N152A and N253A or the equivalent thereof.

[0232] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant neprosin polypeptide comprising an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence corresponding to a prepropolypeptide, propolypeptide, mature polypeptide or polypeptide of SEQ ID NO: 1, wherein the amino acid sequence comprises at least the substitutions N68A, N152A and N253A or the equivalent thereof.

[0233] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant neprosin polypeptide comprising an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence corresponding to a prepropolypeptide, propolypeptide, mature polypeptide or polypeptide of SEQ ID NO: 1, wherein the amino acid sequence comprises at least substitutions N145A, N152A and N253A or the equivalent thereof.

[0234] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant neprosin polypeptide comprising an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence corresponding to a prepropolypeptide, propolypeptide, mature polypeptide or polypeptide of SEQ ID NO: 1, wherein the amino acid sequence comprises at least the substitutions N68A, N145A, N152A and N253A or the equivalent thereof.

[0235] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant neprosin polypeptide comprising an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a polypeptide sequence corresponding to residues 26 to 381 of SEQ ID NO: 44, or a pro- or mature polypeptide thereof, wherein the amino acid at position 68, 152 and / or 254 is not asparagine (N).

[0236] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant neprosin polypeptide comprising an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a polypeptide sequence corresponding to residues 26 to 381 of SEQ ID NO: 46, or a pro- or mature polypeptide thereof, wherein the amino acid at position 68 is not asparagine (N).

[0237] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant neprosin polypeptide comprising an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a polypeptide sequence corresponding to residues 26 to 381 of SEQ ID NO: 48, or a pro- or mature polypeptide thereof, wherein the amino acid at position 152 is not asparagine (N).

[0238] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant neprosin polypeptide comprising an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a polypeptide sequence corresponding to residues 26 to 381 of SEQ ID NO: 50, or a pro- or mature polypeptide thereof, wherein the amino acid at position 254 is not asparagine (N).

[0239] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant neprosin polypeptide comprising an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a polypeptide sequence comprising residues 26 to 381 of SEQ ID NO: 52, or a pro- or mature polypeptide thereof, wherein the amino acids at positions 68 and 254 are not asparagine (N). In some embodiments, the amino acids at positions 68 and 254 are alanine (A).

[0240] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant neprosin polypeptide comprising an amino acid sequence comprising residues 26 to 381 of SEQ ID NO: 44, 46, 48, 50, or 52. In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant neprosin polypeptide comprising a propolypeptide comprising a polypeptide sequence comprising amino acid residues 26 to 381 of SEQ ID NO: 44, 46, 48, 50, or 52. In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant neprosin polypeptide comprising a mature polypeptide comprising a polypeptide sequence comprising amino acid residues 26 to 381 of SEQ ID NO: 44, 46, 48, 50, or 52.

[0241] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant neprosin polypeptide comprising an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence corresponding to residues 105 to 356 of SEQ ID NO: 8, or to a reference sequence corresponding to SEQ ID NO: 8, wherein the amino acid sequence comprises a substitution with an amino acid other than asparagine (N) at at least amino acid position 44, 121, 128 or 229, or a combination thereof, wherein the amino acid positions are relative to SEQ ID NO: 8. In some embodiments, the amino acid substitution or amino acid residue at amino acid position 44, 121, 128 or 229, or a combination thereof, is independently selected from A, C, D, E, F, G, H, I, K, L, M, P, Q, R, S, T, V, W, and Y. In some embodiments, the amino acid substitution or amino acid residue at position 44, 121, 128 or 229, or a combination thereof, is A.

[0242] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant neprosin polypeptide comprising an amino acid sequence comprising residues 105 to 356 of SEQ ID NO: 8, wherein the amino acid sequence comprises a substitution with an amino acid other than asparagine (N) at at least amino acid position 121, 128, or 229, or a combination thereof, wherein the amino acid positions are relative to SEQ ID NO: 8. In some embodiments, the encoded recombinant neprosin polypeptide comprises an amino acid sequence comprising SEQ ID NO: 8, wherein the amino acid sequence comprises a substitution with an amino acid other than asparagine (N) at at least amino acid position 44, 121, 128, or 229, or a combination thereof, wherein the amino acid positions are relative to SEQ ID NO: 8. In some embodiments, the amino acid substitution or amino acid residue at amino acid position 44, 121, 128, or 229, or a combination thereof, is independently selected from A, C, D, E, F, G, H, I, K, L, M, P, Q, R, S, T, V, W, and Y. In some embodiments, the amino acid substitution or amino acid residue at position 44, 121, 128 or 229, or a combination thereof, is A.

[0243] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant neprosin polypeptide comprising an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence corresponding to residues 105 to 356 of SEQ ID NO: 8, or to a reference sequence corresponding to SEQ ID NO: 8, wherein the amino acid sequence comprises a substitution with an amino acid other than asparagine (N) at least at amino acid position 44, wherein the amino acid position is relative to SEQ ID NO: 8. In some embodiments, the amino acid substitution or amino acid residue at position 44 is selected from A, C, D, E, F, G, H, I, K, L, M, P, Q, R, S, T, V, W, and Y. In some embodiments, the amino acid substitution or amino acid residue at position 44 is A.

[0244] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant neprosin polypeptide comprising an acid-activated proteolytically active polypeptide derived from or comprising a subsequence of SEQ ID NO: 10. In some embodiments, the encoded recombinant neprosin polypeptide comprises an amino acid sequence comprising residues 105 to 356 of SEQ ID NO: 10. In some embodiments, the encoded recombinant neprosin polypeptide comprises an amino acid sequence comprising SEQ ID NO: 10.

[0245] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant neprosin polypeptide comprising an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence corresponding to residues 105 to 356 of SEQ ID NO: 8, or to a reference sequence corresponding to SEQ ID NO: 8, wherein the amino acid sequence comprises a substitution with an amino acid other than asparagine (N) at least at amino acid position 121, wherein the amino acid position is relative to SEQ ID NO: 8. In some embodiments, the amino acid substitution or amino acid residue at position 121 is selected from A, C, D, E, F, G, H, I, K, L, M, P, Q, R, S, T, V, W, and Y. In some embodiments, the amino acid substitution or amino acid residue at position 121 is A.

[0246] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant neprosin polypeptide comprising an acid-activated proteolytically active polypeptide derived from or comprising a subsequence of SEQ ID NO: 12. In some embodiments, the encoded recombinant neprosin polypeptide comprises an amino acid sequence comprising residues 105 to 356 of SEQ ID NO: 12. In some embodiments, the encoded recombinant neprosin polypeptide comprises an amino acid sequence comprising SEQ ID NO: 12.

[0247] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant neprosin polypeptide comprising an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence corresponding to residues 105 to 356 of SEQ ID NO: 8, or to a reference sequence corresponding to SEQ ID NO: 8, wherein the amino acid sequence comprises a substitution with an amino acid other than asparagine (N) at least at amino acid position 128, wherein the amino acid position is relative to SEQ ID NO: 8. In some embodiments, the amino acid substitution or amino acid residue at position 128 is selected from A, C, D, E, F, G, H, I, K, L, M, P, Q, R, S, T, V, W, and Y. In some embodiments, the amino acid substitution or amino acid residue at position 128 is A.

[0248] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant neprosin polypeptide comprising an acid-activated proteolytically active polypeptide derived from or comprising a subsequence of SEQ ID NO: 14. In some embodiments, the encoded recombinant neprosin polypeptide comprises an amino acid sequence comprising residues 105 to 356 of SEQ ID NO: 14. In some embodiments, the encoded recombinant neprosin polypeptide comprises an amino acid sequence comprising SEQ ID NO: 14.

[0249] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant neprosin polypeptide comprising an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence corresponding to residues 105 to 356 of SEQ ID NO: 8, or to a reference sequence corresponding to SEQ ID NO: 8, wherein the amino acid sequence comprises a substitution with an amino acid other than asparagine (N) at least at amino acid position 229, wherein the amino acid position is relative to SEQ ID NO: 8. In some embodiments, the amino acid substitution or amino acid residue at position 229 is selected from A, C, D, E, F, G, H, I, K, L, M, P, Q, R, S, T, V, W, and Y. In some embodiments, the amino acid substitution or amino acid residue at position 229 is A.

[0250] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant neprosin polypeptide comprising an acid-activated proteolytically active polypeptide derived from or comprising a subsequence of SEQ ID NO: 16. In some embodiments, the encoded recombinant neprosin polypeptide comprises an amino acid sequence comprising residues 105 to 356 of SEQ ID NO: 16. In some embodiments, the encoded recombinant neprosin polypeptide comprises an amino acid sequence comprising SEQ ID NO: 16.

[0251] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant neprosin polypeptide comprising an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence corresponding to residues 105 to 356 of SEQ ID NO: 8, or to a reference sequence corresponding to SEQ ID NO: 8, wherein the amino acid sequence comprises a substitution with an amino acid other than asparagine (N) at at least amino acid positions 121 and 229, wherein the amino acid positions are relative to SEQ ID NO: 8. In some embodiments, the amino acid substitution or amino acid residue at each of positions 121 and 229 is independently selected from A, C, D, E, F, G, H, I, K, L, M, P, Q, R, S, T, V, W, and Y. In some embodiments, the amino acid substitution or amino acid residue at amino acid positions 121 and 229 is A.

[0252] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant neprosin polypeptide comprising an acid-activated proteolytically active polypeptide derived from or comprising a subsequence of SEQ ID NO: 18. In some embodiments, the encoded recombinant neprosin polypeptide comprises an amino acid sequence comprising residues 105 to 356 of SEQ ID NO: 18. In some embodiments, the encoded recombinant neprosin polypeptide comprises an amino acid sequence comprising SEQ ID NO: 18.

[0253] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant neprosin polypeptide comprising an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence corresponding to residues 105 to 356 of SEQ ID NO: 8, or to a reference sequence corresponding to SEQ ID NO: 8, wherein the amino acid sequence comprises a substitution with an amino acid other than asparagine (N) at at least amino acid positions 44, 128, and 229, wherein the amino acid positions are relative to SEQ ID NO: 8. In some embodiments, the amino acid substitution or amino acid residue at each of positions 44, 128, and 229 is independently selected from A, C, D, E, F, G, H, I, K, L, M, P, Q, R, S, T, V, W, and Y. In some embodiments, the amino acid substitution or amino acid residue at amino acid positions 44, 128, and 229 is A.

[0254] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant neprosin polypeptide comprising an acid-activated proteolytically active polypeptide derived from or comprising a subsequence of SEQ ID NO: 20. In some embodiments, the encoded recombinant neprosin polypeptide comprises an amino acid sequence comprising residues 105 to 356 of SEQ ID NO: 20. In some embodiments, the encoded recombinant neprosin polypeptide comprises an amino acid sequence comprising SEQ ID NO: 20.

[0255] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant neprosin polypeptide comprising an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence corresponding to residues 105 to 356 of SEQ ID NO: 8, or to a reference sequence corresponding to SEQ ID NO: 8, wherein the amino acid sequence comprises a substitution with an amino acid other than asparagine (N) at at least amino acid positions 44, 121, 128, and 229, wherein the amino acid positions are relative to SEQ ID NO: 8. In some embodiments, the amino acid substitution or amino acid residue at each of positions 44, 121, 128, and 229 is independently selected from A, C, D, E, F, G, H, I, K, L, M, P, Q, R, S, T, V, W, and Y. In some embodiments, the amino acid substitution or amino acid residue at amino acid positions 44, 121, 128, and 229 is A.

[0256] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant neprosin polypeptide comprising an acid-activated proteolytically active polypeptide derived from or comprising a subsequence of SEQ ID NO: 22. In some embodiments, the encoded recombinant neprosin polypeptide comprises an amino acid sequence comprising residues 105 to 356 of SEQ ID NO: 22. In some embodiments, the encoded recombinant neprosin polypeptide comprises an amino acid sequence comprising SEQ ID NO: 22.

[0257] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant neprosin polypeptide comprising an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence corresponding to residues 105 to 356 of SEQ ID NO: 8, or to a reference sequence corresponding to SEQ ID NO: 8, wherein the amino acid sequence comprises a substitution with an amino acid other than asparagine (N) at at least amino acid positions 44 and 229, wherein the amino acid positions are relative to SEQ ID NO: 8. In some embodiments, the amino acid substitution or amino acid residue at each of positions 44 and 229 is independently selected from A, C, D, E, F, G, H, I, K, L, M, P, Q, R, S, T, V, W, and Y. In some embodiments, the amino acid substitution or amino acid residue at amino acid positions 44 and 229 is A.

[0258] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant neprosin polypeptide comprising an acid-activated proteolytically active polypeptide derived from or comprising a subsequence of SEQ ID NO: 32. In some embodiments, the encoded recombinant neprosin polypeptide comprises an amino acid sequence comprising residues 105 to 356 of SEQ ID NO: 32. In some embodiments, the encoded recombinant neprosin polypeptide comprises an amino acid sequence comprising SEQ ID NO: 32.

[0259] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant neprosin polypeptide comprising an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the sequence corresponding to residues 105 to 356 of SEQ ID NO: 8, or to the sequence corresponding to SEQ ID NO: 8.

[0260] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant neprosin polypeptide comprising an acid-activated proteolytically active polypeptide derived from or comprising a subsequence of SEQ ID NO: 8. In some embodiments, the encoded recombinant neprosin polypeptide comprises an amino acid sequence comprising residues 105 to 356 of SEQ ID NO: 8. In some embodiments, the encoded recombinant neprosin polypeptide comprises an amino acid sequence comprising SEQ ID NO: 8.

[0261] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant neprosin polypeptide comprising an amino acid sequence comprising residues 96 to 451 of SEQ ID NO: 58, 60, 62, 64, 66, 68, or 70. In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant neprosin polypeptide comprising a propolypeptide comprising an amino acid sequence comprising amino acid residues 96 to 451 of SEQ ID NO: 58, 60, 62, 64, 66, 68, or 70. In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant neprosin polypeptide comprising a mature polypeptide comprising an amino acid sequence comprising amino acid residues 96 to 451 of SEQ ID NO: 58, 60, 62, 64, 66, 68, or 70.

[0262] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more sequence identity to a reference polynucleotide sequence corresponding to nucleotide residues 313 to 1068 of SEQ ID NO: 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, or 33, or a polynucleotide sequence corresponding to SEQ ID NO: 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, or 33, wherein the recombinant polynucleotide encodes a neprosin polypeptide.

[0263] In some embodiments, the recombinant polynucleotide comprises nucleotide residues 313 to 1068 comprising SEQ ID NO: 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, or 33, or a polynucleotide sequence comprising SEQ ID NO: 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, or 33.

[0264] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant neprosin polypeptide comprising a signal sequence or signal peptide as discussed herein. In some embodiments, the encoded signal sequence or signal peptide is functional in a host cell used or to be used for expressing a neprosin polypeptide. In some embodiments, the encoded signal sequence or signal peptide is fused to a propolypeptide form of recombinant neprosin to form a pre-propolypeptide. In some embodiments, the encoded signal sequence or signal peptide is fused to a polypeptide comprising a mature active form of recombinant neprosin. In some embodiments, the encoded signal sequence may be a naturally occurring signal sequence or a synthetic signal sequence, including a hybrid signal sequence.

[0265] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant neprosin polypeptide containing a fusion protein. In some embodiments, the encoded recombinant neprosin polypeptide can be fused with a variety of polypeptide sequences as described above. In some embodiments, the fusion protein of the recombinant neprosin polypeptide comprises a glycine-histidine tag or a histidine tag (His tag). In some embodiments, the fusion protein of the recombinant neprosin polypeptide comprises an epitope tag, such as c-myc, FLAG, V5 or hemagglutinin (HA). In some embodiments, the fusion protein of the recombinant neprosin polypeptide comprises a GST, SUMO, Strep, MBP or GFP tag.

[0266] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant neprosin polypeptide comprising a signal sequence and / or a signal peptide and a polypeptide tag. In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant neprosin polypeptide comprising SEQ ID NO: 36, 38, 40, 42, 44, 46, 48, 50 or 52. In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant neprosin polypeptide comprising SEQ ID NO: 56, 58, 60, 62, 64, 66, 68 or 70.

[0267] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence comprising SEQ ID NO: 35, 37, 39, 41, 43, 45, 47, 49, or 51. In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence comprising SEQ ID NO: 55, 57, 59, 61, 63, 65, 67, or 69 encoding a recombinant neprosin polypeptide.

[0268] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant neprosin polypeptide comprising SEQ ID NO: 1, 2, 3, 4, 5, or 6.

[0269] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant neprosin polypeptide comprising an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a sequence corresponding to a prepropolypeptide, propolypeptide, mature polypeptide or polypeptide of SEQ ID NO: 1, 2, 3, 4, 5 or 6.

[0270] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant neprosin polypeptide comprising an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the sequence corresponding to the prepropolypeptide, propolypeptide, mature polypeptide or polypeptide of SEQ ID NO: 1.

[0271] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant neprosin polypeptide comprising an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the sequence of the propolypeptide corresponding to SEQ ID NO: 1.

[0272] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant neprosin polypeptide comprising an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the sequence corresponding to the mature polypeptide of SEQ ID NO: 1.

[0273] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant neprosin polypeptide comprising an amino acid sequence of a pre-propeptide comprising SEQ ID NO: 1. In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant neprosin polypeptide comprising an amino acid sequence of a pro-polypeptide comprising SEQ ID NO: 1. In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant neprosin polypeptide comprising an amino acid sequence of a mature polypeptide comprising SEQ ID NO: 1. In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant neprosin polypeptide comprising an acid-activated proteolytically active polypeptide derived from a subsequence of a sequence corresponding to SEQ ID NO: 1 or comprising a subsequence of a sequence corresponding to SEQ ID NO: 1.

[0274] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant neprosin polypeptide comprising an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the sequence corresponding to the prepropolypeptide, propolypeptide, mature polypeptide or polypeptide of SEQ ID NO: 2.

[0275] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant neprosin polypeptide comprising an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the sequence of the propolypeptide corresponding to SEQ ID NO: 2.

[0276] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant neprosin polypeptide comprising an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the sequence corresponding to the mature polypeptide of SEQ ID NO: 2.

[0277] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant neprosin polypeptide comprising an amino acid sequence of a pre-propeptide comprising SEQ ID NO: 2. In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant neprosin polypeptide comprising an amino acid sequence of a pro-polypeptide comprising SEQ ID NO: 2. In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant neprosin polypeptide comprising an amino acid sequence of a mature polypeptide comprising SEQ ID NO: 2. In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant neprosin polypeptide comprising an acid-activated proteolytically active polypeptide derived from a subsequence of a sequence corresponding to SEQ ID NO: 2 or comprising a subsequence of a sequence corresponding to SEQ ID NO: 2.

[0278] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant neprosin polypeptide comprising an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the sequence corresponding to the prepropolypeptide, propolypeptide, mature polypeptide or polypeptide of SEQ ID NO: 3.

[0279] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant neprosin polypeptide comprising an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the sequence of the propolypeptide corresponding to SEQ ID NO: 3.

[0280] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant neprosin polypeptide comprising an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the sequence corresponding to the mature polypeptide of SEQ ID NO: 3.

[0281] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant neprosin polypeptide comprising an amino acid sequence of a prepropolypeptide comprising SEQ ID NO: 3. In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant neprosin polypeptide comprising an amino acid sequence of a propolypeptide comprising SEQ ID NO: 3. In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant neprosin polypeptide comprising an amino acid sequence of a mature polypeptide comprising SEQ ID NO: 3. In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant neprosin polypeptide comprising an acid-activated proteolytically active polypeptide derived from a subsequence of a sequence corresponding to SEQ ID NO: 3 or comprising a subsequence of a sequence corresponding to SEQ ID NO: 3.

[0282] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant neprosin polypeptide comprising an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the sequence corresponding to the prepropolypeptide, propolypeptide, mature polypeptide or polypeptide of SEQ ID NO: 4.

[0283] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant neprosin polypeptide comprising an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the sequence of the propolypeptide corresponding to SEQ ID NO: 4.

[0284] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant neprosin polypeptide comprising an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the sequence corresponding to the mature polypeptide of SEQ ID NO: 4.

[0285] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant neprosin polypeptide comprising an amino acid sequence of a prepropolypeptide comprising SEQ ID NO: 4. In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant neprosin polypeptide comprising an amino acid sequence of a propolypeptide comprising SEQ ID NO: 4. In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant neprosin polypeptide comprising an amino acid sequence of a mature polypeptide comprising SEQ ID NO: 4. In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant neprosin polypeptide comprising an acid-activated proteolytically active polypeptide derived from a subsequence of a sequence corresponding to SEQ ID NO: 4 or comprising a subsequence of a sequence corresponding to SEQ ID NO: 4.

[0286] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant neprosin polypeptide comprising an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the sequence corresponding to the prepropolypeptide, propolypeptide, mature polypeptide or polypeptide of SEQ ID NO: 5.

[0287] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant neprosin polypeptide comprising an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the sequence of the propolypeptide corresponding to SEQ ID NO: 5.

[0288] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant neprosin polypeptide comprising an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the sequence corresponding to the mature polypeptide of SEQ ID NO: 5.

[0289] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant neprosin polypeptide comprising an amino acid sequence of a prepropolypeptide comprising SEQ ID NO: 5. In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant neprosin polypeptide comprising an amino acid sequence of a propolypeptide comprising SEQ ID NO: 5. In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant neprosin polypeptide comprising an amino acid sequence of a mature polypeptide comprising SEQ ID NO: 5. In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant neprosin polypeptide comprising an acid-activated proteolytically active polypeptide derived from a subsequence of a sequence corresponding to SEQ ID NO: 5 or comprising a subsequence of a sequence corresponding to SEQ ID NO: 5.

[0290] In some embodiments, the recombinant polynucleotide encodes a recombinant neprosin polypeptide comprising an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the sequence corresponding to the prepropolypeptide, propolypeptide, mature polypeptide or polypeptide of SEQ ID NO: 6.

[0291] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant neprosin polypeptide comprising an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the sequence of the propolypeptide corresponding to SEQ ID NO: 6.

[0292] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant neprosin polypeptide comprising an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the sequence corresponding to the mature polypeptide of SEQ ID NO: 6.

[0293] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant neprosin polypeptide comprising an amino acid sequence of a prepropolypeptide comprising SEQ ID NO: 6. In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant neprosin polypeptide comprising an amino acid sequence of a propolypeptide comprising SEQ ID NO: 6. In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant neprosin polypeptide comprising an amino acid sequence of a mature polypeptide comprising SEQ ID NO: 6. In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant neprosin polypeptide comprising an acid-activated proteolytically active polypeptide derived from a subsequence of a sequence corresponding to SEQ ID NO: 6 or comprising a subsequence of a sequence corresponding to SEQ ID NO: 6.

[0294] On the other hand, the disclosure also provides expression vectors comprising recombinant polynucleotides encoding recombinant neprosin polypeptides described herein, for example, for expressing encoded recombinant neprosin polypeptides. In some embodiments, the expression vector comprises one or more control sequences to regulate the expression of neprosin polynucleotides and / or polypeptides. In some embodiments, the control sequences include promoters, leader sequences, polyadenylation sequences, propeptide sequences, signal peptide sequences, and transcription terminators, among others. In particular, control sequences such as promoters, leader sequences, polyadenylation sequences, propeptide sequences, signal peptide sequences, and transcription terminators are those that are effective in selected host cells (e.g., mammalian cells, insect cells, or fungal cells).

[0295] In some embodiments, suitable promoters for guiding the transcription of nucleic acid constructs in mammalian cells include SV40 promoters or promoters from cytomegalovirus (CMV), chicken β-actin (CAG) or elongation factor (EF) -1, and others. In some embodiments, suitable promoters for guiding the transcription of nucleic acid constructs in insect cells include baculovirus promoters (e.g., P10 and polyhedron promoters), OpIE2 promoters and black-tailed leafhopper (Nephotettixcincticeps) actin promoters, and others. In some embodiments, suitable promoters for guiding transcription in fungal cells include promoters of AOX1 gene, TEF1 gene, POX2 gene, TDH3 gene, PGK gene, ADH1 gene and hybrid promoters (see, e.g., Mojzita et al., Curr Opin Biotech., 2019, 59: 141-149; Yang et al., World Journal of Microbiology and Biotechnology, 2015, 31: 1641-1646), and others. Other fungal promoters include promoters from the genes for Aspergillus oryzae TAKA amylase, Rhizomucor miehei aspartic proteinase, Aspergillus niger neutral alpha-amylase, Aspergillus niger acid-stable alpha-amylase, Aspergillus niger or Aspergillus awamori glucoamylase (glaA), Rhizomucor miehei lipase, Aspergillus oryzae alkaline protease, Aspergillus oryzae triose phosphate isomerase, Aspergillus nidulans acetamidase, and Fusarium oxysporum trypsin-like protease (see, e.g., WO 96 / 00787), as well as the NA2-tpi promoter (a hybrid of the promoters from the Aspergillus niger neutral alpha-amylase gene and the Aspergillus oryzae triose phosphate isomerase gene), and mutants, truncated, and hybrid promoters thereof. Exemplary yeast cell promoters can be derived from the genes for Saccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiae galactokinase (GAL1), Saccharomyces cerevisiae alcohol dehydrogenase / glyceraldehyde-3-phosphate dehydrogenase (ADH2 / GAP), and Saccharomyces cerevisiae 3-phosphoglycerate kinase.

[0296] In some embodiments, the control sequence is also a suitable transcription terminator sequence (i.e., a sequence recognized by the host cell to terminate transcription). In some embodiments, the terminator sequence is operably linked to the 3' end of the nucleic acid sequence encoding the neprosin polypeptide. Any suitable terminator that is functional in the selected host cell can be used for the expression of the neprosin polypeptide. For example, but not limited to, transcription terminators include those from human β-globin gene, SV40, hGH, IE1 terminator and BGH. Exemplary fungal transcription terminators include those from the following genes: Aspergillus oryzae TAKA amylase, Aspergillus niger glucoamylase, Aspergillus nidulans anthranilate synthase, Aspergillus niger α-glucosidase and Fusarium oxysporum trypsin-like protease, and others. Exemplary terminators for yeast host cells can be obtained from the following genes: Saccharomyces cerevisiae enolase, Saccharomyces cerevisiae cytochrome C (CYC1), and Saccharomyces cerevisiae glyceraldehyde-3-phosphate dehydrogenase.

[0297] In some embodiments, the control sequence is also a suitable leader sequence (i.e., an untranslated region of an mRNA important for translation of a host cell). In some embodiments, the leader sequence is operably linked to the 5' end of the nucleic acid sequence encoding the recombinant neprosin polypeptide. Any suitable leader sequence that is functional in a selected host cell can be used for expression of the recombinant neprosin polypeptide. Exemplary leaders of mammalian and insect cells include leaders of expression genes (e.g., heat shock proteins, myosin, BIP immunoglobulin binding protein, GRP glucose-regulated protein, etc.), viral leaders (e.g., EMC virus), and synthetic leaders (e.g., hTEE-658), and, for example, Cao et al., Nature Commun., 2021, 12: Those described in 4138, and others, incorporated herein by reference. Exemplary leaders for fungal expression include those from Aspergillus oryzae TAKA amylase and Aspergillus nidulans triose phosphate isomerase, and others. Suitable leaders for yeast host cells are obtained from the genes for Saccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiae 3-phosphoglycerate kinase, Saccharomyces cerevisiae alpha-factor, and Saccharomyces cerevisiae alcohol dehydrogenase / glyceraldehyde-3-phosphate dehydrogenase (ADH2 / GAP).

[0298] In some embodiments, the control sequence is also a polyadenylation sequence (i.e., a sequence operably linked to the 3' end of the nucleic acid sequence, and which is recognized by the host cell as a signal to add polyadenosine residues to the transcribed mRNA when transcribed). Any suitable polyadenylation sequence that is functional in the selected host cell can be used in the present invention. Exemplary polyadenylation sequences for mammalian and insect cells include those of the genes for human and mouse α-globin, mouse κ light chain, chicken ovalbumin, SV40, and synthetic poly A sequences (see, e.g., Clerici et al., eLife, 2017, 6: e33111), as well as others. Exemplary polyadenylation sequences for fungal host cells include, but are not limited to, genes for: Aspergillus oryzae TAKA amylase, Aspergillus niger glucoamylase, Aspergillus nidulans anthranilate synthase, Fusarium oxysporum trypsin-like protease, and Aspergillus niger α-glucosidase. Useful polyadenylation sequences for yeast host cells are known in the art (see, eg, Guo and Sherman, Mol. Cell. Bio., 1995, 15:5983-5990).

[0299] In some embodiments, the control sequence is also a signal peptide (i.e., a coding region that encodes an amino acid sequence that is connected to the amino terminus of a polypeptide and directs the encoded polypeptide to the secretory pathway of a cell), as described herein. In some embodiments, the 5' end of the coding sequence of the nucleic acid sequence inherently comprises a signal peptide coding region that is naturally connected to the segment of the coding region encoding the secretory polypeptide in the translation reading frame. Alternatively, in some embodiments, the 5' end of the coding sequence contains a signal peptide coding region that is foreign to the coding sequence, i.e., a heterologous signal sequence. Any suitable signal peptide coding region that directs the expressed polypeptide to the secretory pathway of a selected host cell can be used for the expression of engineered polypeptides. In some embodiments, the signal sequence is a mammalian cell signal sequence. In some embodiments, the signal sequence is an insect cell signal sequence. In some embodiments, the signal sequence is a fungal cell signal sequence. Exemplary signal sequences are described herein.

[0300] In another aspect, the present disclosure provides an expression vector comprising a recombinant polynucleotide encoding a recombinant neprosin polypeptide and one or more expression regulatory regions such as a promoter and terminator, an origin of replication, etc. (depending on the type of host into which it is to be introduced). In some embodiments, the various nucleic acids and control sequences described herein are linked together (i.e., operably linked) to produce a recombinant expression vector comprising one or more convenient restriction sites to allow insertion or substitution of the nucleic acid sequence encoding the recombinant neprosin polypeptide at these sites.

[0301] The recombinant expression vector can be any suitable vector (e.g., plasmid or virus) that can be easily subjected to recombinant DNA procedures and cause the expression of the polynucleotide sequence encoding neprosin. The choice of vector generally depends on the compatibility of the vector with the host cell into which the vector is to be introduced. The vector can be a linear plasmid or a closed circular plasmid.

[0302] In some embodiments, the expression vector is an autonomously replicating vector (i.e., a vector that exists as an extrachromosomal entity, whose replication is independent of chromosomal replication, such as a plasmid, an extrachromosomal element, a minichromosome, or an artificial chromosome). The vector may include any means for ensuring self-replication. In some selectable embodiments, the vector is a vector that, when introduced into a host cell, is integrated into the genome and replicates with the chromosome into which it is integrated. In addition, in some embodiments, a single vector or plasmid or two or more vectors or plasmids that together contain all the DNA to be introduced into the genome of the host cell, and / or a transposon are utilized.

[0303] In some embodiments, expression vector comprises one or more selectable markers, and it allows to easily select transformed cells." selectable marker " is that its product provides biocide or virus tolerance, for the tolerance of heavy metals, provides the gene of prototrophy etc. to auxotrophy.According to cell type, suitable markers for mammalian cells include adenosine deaminase (ADA), aminoglycoside phosphotransferase (neo, G418, APH), bleomycin (Sh ble), cytosine deaminase, dihydrofolate reductase (DHFR), histidinol dehydrogenase (hisD), hygromycin-B-phosphotransferase (HPH), thymidine kinase (TK) and xanthine-guanine phosphoribosyltransferase (XGPRT, gpt), and other.Depend on cell type, suitable markers for insect cells include puromycin acetyltransferase, aminoglycoside phosphotransferase and hygromycin-B-phosphotransferase (hygromycin resistance), and other. Suitable markers for fungal host cells include, but are not limited to, ADE2, HIS3, LEU2, LYS2, MET3, TRP1, and URA3. Selectable markers for use in filamentous fungal host cells include, but are not limited to, amdS (acetamidase; e.g., from Aspergillus nidulans or Aspergillus oryzae), argB (ornithine carbamoyltransferase), bar (phosphinothricin acetyltransferase; e.g., from S. hygroscopicus), hph (hygromycin phosphotransferase), niaD (nitrate reductase), pyrG (orotidine-5'-phosphate decarboxylase; e.g., from Aspergillus nidulans or Aspergillus oryzae), sC (sulfate adenyltransferase), and trpC (anthranilate synthase), and equivalents thereof.

[0304] In another aspect, the disclosure provides a host cell comprising an expression vector described herein, wherein the host cell is a eukaryotic cell. In some embodiments, the host cell is a mammalian cell, an insect cell, or a fungal cell.

[0305] In some embodiments, the host cell expresses a recombinant neprosin polypeptide described herein. In some embodiments, the recombinant neprosin polypeptide expressed in the host cell is a pre-propolypeptide or pre-zymogen form of the neprosin polypeptide. In some embodiments, the recombinant neprosin polypeptide expressed in the host cell is a propolypeptide or zymogen form of the neprosin polypeptide. In some embodiments, the recombinant neprosin polypeptide expressed in the host cell is a mature neprosin polypeptide.

[0306] In some embodiments, the host cell comprises a polynucleotide sequence encoding a recombinant neprosin polypeptide, wherein the encoded recombinant neprosin polypeptide comprises a signal sequence. In some embodiments, a signal sequence suitable for use in the host cell is selected. In some embodiments, the signal sequence is a mouse signal sequence. In some embodiments, the signal sequence is a human signal sequence. In some embodiments, the signal sequence is an insect cell signal sequence. In some embodiments, the signal sequence is a fungal signal sequence. In some embodiments, the host cell expresses a secreted recombinant neprosin polypeptide. In some embodiments, the host cell expresses a soluble form of the recombinant neprosin polypeptide.

[0307] In some embodiments, the host cell expresses a glycosylated recombinant neprosin polypeptide. In some embodiments, the host cell expresses a recombinant neprosin polypeptide that is glycosylated but lacks glycosylation at at least one glycosylation site, as described herein. In some embodiments, the host cell expresses a non-glycosylated recombinant neprosin polypeptide. In some embodiments, the glycosylation pattern of the recombinant neprosin polypeptide is manipulated by appropriate selection of the host cell.

[0308] In some embodiments, the host cell is a mammalian cell. In some embodiments, the host cell is a human cell or a rodent cell. Exemplary mammalian cells include Expi293, HeLa, U2OS, A549, HT1080, CAD, P19, NIH 3T3, L929, Hek 293, 293F, 293E, 293T, COS, Vero, NS0, Sp2 / 0 cells, DUKX-X11, MCF-7, Y79, SO-Rb50, Hep G2, J558L and CHO cells, and others. In some embodiments, the mammalian host cell is defective in glycosylation (see, e.g., Esko et al., Glycosylation Mutants of Cultured Mammalian Cells. 2017. In: Varki A, Cummings RD, Esko JD et al., eds. Essentials of Glycobiology, 3rd ed., Chapter 49, Cold Spring Harbor (NY): Cold Spring Harbor Laboratory Press; 2015-2017). In some embodiments, the polynucleotide sequence encoding the recombinant neprosin polypeptide in the host mammalian cell is codon-optimized for expression in mammalian cells.

[0309] In some embodiments, the host cell is an insect cell. In some embodiments, the insect host cell is a Lepidoptera or Diptera insect cell. Exemplary insect host cells include Sf9 cells, Sf21 cells, Schneider 2 cells and BTI-TN-5B1-4 (High Five) cells, and others. In some embodiments, the insect host cell contains humanized glycosylation pathway enzymes (see, e.g., Yee et al., Ind. Eng. Chem. Res., 2018, 57: 10061-10070). In some embodiments, the polynucleotide sequence encoding the recombinant neprosin polypeptide in the host insect cell is optimized for expression of codons in insect cells.

[0310] In some embodiments, the host cell is a fungal cell. In some embodiments, the fungal cell is a filamentous fungal cell or a fungus. In some embodiments, the fungal cell is a yeast cell. In some embodiments, the fungal host cell is a Pichia, Saccharomyces, Yarrowia, Kluyveromyces, Aspergillus, Trichoderma, Neurospora, Mucor, Penicillium, Trichoderma or Myceliophthora fungal cell. Exemplary fungal host cells include Pichia pastoris, Yarrowia lipolytica, Kluyveromyces marxianus, Kluyveromyces lactis, Aspergillus niger, Aspergillus oryzae, Aspergillus fumigatus, Trichoderma reesei, Neurospora crassa, Mucorcircinelloides, Penicillium chrysogenum, T. reesei, Trichoderma harzianum, Saccharomyces cerevisiae, and Myceliophthora thermophile, among others. In some embodiments, the polynucleotide sequence encoding the recombinant neprosin polypeptide in the host fungal cell is codon-optimized for expression in the fungal cell. In a preferred embodiment, the fungal cell used to express the recombinant neprosin polypeptide is Pichia pastoris or Saccharomyces cerevisiae.

[0311] In some embodiments, any suitable method for introducing a polynucleotide for expressing a recombinant neprosin polypeptide into a cell can be used for purposes herein. Suitable techniques include, but are not limited to, electroporation, biolistic particle bombardment, liposome-mediated transfection, calcium chloride transfection, and protoplast fusion. Expression and processing of recombinant neprosin polypeptide

[0312] In another aspect, the disclosure provides a method of expressing a recombinant neprosin polypeptide described herein. In some embodiments, the disclosure also provides processing of the expressed recombinant neprosin polypeptide to prepare a proteolytically active recombinant neprosin polypeptide, particularly a proteolytically active form of a mature neprosin polypeptide.

[0313] In some embodiments, the method of producing a recombinant neprosin polypeptide comprises culturing a host cell comprising an expression vector comprising a recombinant polynucleotide encoding a recombinant neprosin polypeptide described herein under suitable culture conditions such that the encoded recombinant neprosin polypeptide is expressed or produced. In some embodiments, the host cell is a mammalian cell, an insect cell, or a fungal cell.

[0314] In some embodiments, the method of producing a recombinant neprosin polypeptide comprises culturing a mammalian cell comprising an expression vector comprising a recombinant polynucleotide encoding a recombinant neprosin polypeptide described herein under suitable culture conditions such that the encoded recombinant neprosin polypeptide is expressed or produced.

[0315] As described herein, in some embodiments, mammalian cells are rodent cells or human cells. Exemplary mammalian cells used as host cells include Expi293, HeLa, U2OS, A549, HT1080, CAD, P19, NIH3T3, L929, Hek 293, 293F, 293E, 293T, COS, Vero, NS0 cells, Sp2 / 0, DUKX-X11, MCF-7, Y79, SO-Rb50, Hep G2, J558L and CHO cells. In some embodiments, the mammalian cell is defective in glycosylation (see, e.g., Esko et al., Glycosylation Mutants of Cultured Mammalian Cells. 2017. In: Varki A, Cummings RD, Esko JD et al., eds. Essentials of Glycobiology, 3rd ed., Chapter 49, Cold Spring Harbor (NY): Cold Spring Harbor Laboratory Press; 2015-2017).

[0316] In some embodiments, the method of producing a recombinant neprosin polypeptide comprises culturing an insect cell comprising an expression vector comprising a recombinant polynucleotide encoding a recombinant neprosin polypeptide described herein under suitable culture conditions such that the encoded recombinant neprosin polypeptide is expressed or produced.

[0317] As described above, in some embodiments, the insect cell is a Lepidoptera or Diptera insect cell. Exemplary insect cells used as host cells include Sf9 cells, Sf21 cells, BTI-TN-5B1-4 (High Five) cells, and Schneider 2 cells. In some embodiments, the insect cell contains humanized glycosylation pathway enzymes (see, e.g., Yee et al., Ind. Eng. Chem. Res., 2018, 57: 10061-10070).

[0318] In some embodiments, a method of producing a recombinant neprosin polypeptide comprises culturing a fungal cell comprising an expression vector comprising a recombinant polynucleotide encoding a recombinant neprosin polypeptide described herein under suitable culture conditions such that the encoded recombinant neprosin polypeptide is expressed or produced.

[0319] As described above, in some embodiments, the fungal cell is a filamentous fungal cell or a fungus. In some embodiments, the fungal cell is a yeast cell. Exemplary fungal cells for use as host cells include Aspergillus, Arxula, Aurantiochytrium, Candida, Claviceps, Cryptococcus, Cunninghamella, Geotrichum, Hansenula, Kluyveromyces, Kodamaea, Komagataella, Leucosporidiella, Lipomyces, Morterella, Mucor, Myceliophthora, Neurospora, Ogataea, Penicillium, Pichia, Prototheca, Rhizopus, Rhodosporidium, Rhodotorula, Saccharomyces, Tremella, Trichoderma, and Yarrowia fungal cells. In some embodiments, the fungal cell used as a host cell is Pichia pastoris, Yarrowia lipolytica, Kluyveromyces marxianus, Kluyveromyces lactis, Aspergillus niger, Aspergillus oryzae, Aspergillus fumigatus, Trichoderma reesei, Neurospora crassa, Mucor circinelloides, Penicillium chrysogenum, Trichoderma reesei, Trichoderma harzianum, Saccharomyces cerevisiae, and Myceliophthora thermophila.

[0320] Suitable culture media and growth conditions for mammalian, insect and fungal cells expressing heterologous proteins are well known in the art. Exemplary culture media for mammalian cells include Eagle's Minimal Essential Medium (EMEM), Dulbecco's Modified Eagle's Medium (DMEM), Iscove's Modified Dulbecco's Medium (IMDM), Hybri-Care Medium, McCoy's 5A and RPMI-1640, Ham's Nutrient Mixture and DMEM / F12 medium, among others. Such media preparations are described in the ATCC Animal Cell Culture Guide, 2022; Phelan, K. and May, KM, 2017, “Mammalian cell tissue culture techniques,” Curr. Protoc. Mol. Biol. 117:A.3F.1-A.3F.23; and Freshney, IF, "Culture of Animal Cells: A Manual of Basic Technique and Specialized Applications,” 7th Edition, Wiley-Blackwell (2016), among others.

[0321] Exemplary culture media for insect cells include Grace insect medium, complete TNM-FH, Sf 900 II serum-free medium, Express Five serum-free medium, Mitsuhashi and Maramorosh basal medium, HH70 medium, and Schneider fruit fly medium, among others. Such culture medium preparations are described in Agathos, SN "Development of serum-free media for lepidopteran insect cell lines", Baculovirus Expression Protocols, 2nd edition, DW Murhammer ed., pp. 155-185, Humana Press, Totowa, NJ (2007); and Käßer et al., Electronic Journal of Biotechnology, 2022, 56: 54-64, among others.

[0322] The culture medium for fungal cells can vary according to the type of fungal cells for expressing recombinant neprosin polypeptides. Exemplary culture medium for Pichia pastoris is Rich defined culture medium (RDM) or buffered composite glycerol medium (BMGY) (see Mathews et al., Biotechnol Bioeng, 2018, 115 (1): 103-113); YNB / YND culture medium (yeast nitrogen source basis), YPM culture medium or BMM (buffered minimal medium) (see, e.g., Lin-Cereghino et al., "Expression of protein in Pichia pastoris", In Expression systems: methods express., Dyson M, Durocher Y (ed), pp. 123-145, Scion Publishing, Banbury, United Kingdom (2007)). Exemplary culture medium for saccharomyces cerevisiae includes YPD and YEPD culture medium (see, e.g., Dymond, JS., Methods Enzymol., 2013, 533: 191-204), and others.

[0323] In some embodiments, the method further comprises isolating the expressed recombinant neprosin polypeptide. In some embodiments, the method further comprises the step of purifying the expressed recombinant neprosin polypeptide. In some embodiments, the isolated or purified neprosin polypeptide comprises a pre-propolypeptide (e.g., pre-zymogen) form of the recombinant neprosin polypeptide. In some embodiments, the isolated or purified neprosin polypeptide comprises a propolypeptide (e.g., zymogen) form of the recombinant neprosin polypeptide. In some embodiments, the isolated or purified neprosin polypeptide comprises a mature form of the recombinant neprosin polypeptide.

[0324] In some embodiments, recombinant neprosin polypeptides expressed or produced in host cells are isolated and / or purified from cells and / or culture medium using any one or more known techniques for protein purification, including detergent treatment, sonication, filtration, salting out, ultracentrifugation, and chromatography, among others. Chromatographic techniques for separating and purifying neprosin polypeptides include reverse phase chromatography, high performance liquid chromatography, ion exchange chromatography, hydrophobic interaction chromatography, size exclusion chromatography, gel electrophoresis, and affinity chromatography, among others. The conditions for purifying a particular enzyme depend in part on factors such as net charge, hydrophobicity, hydrophilicity, molecular weight, molecular shape, and the like, and will be apparent to one skilled in the art. In some embodiments, affinity techniques can be used to separate recombinant neprosin polypeptides. For affinity chromatography purification, antibodies that specifically bind to the neprosin polypeptide of interest can be used. In some embodiments, the protein is separated and / or purified using a polypeptide tag (e.g., a His tag) present in the recombinant neprosin polypeptide.

[0325] In some embodiments, the method is used to express a pre-propolypeptide (e.g., pre-zymogen) form of a recombinant or naturally occurring neprosin polypeptide. In some embodiments, the method is used to express a propolypeptide, e.g., zymogen form, of a recombinant or naturally occurring neprosin polypeptide. In some embodiments, the method is used to express a mature polypeptide form of a recombinant or naturally occurring neprosin polypeptide.

[0326] In some embodiments, the method is used to express a secreted recombinant neprosin polypeptide. In some embodiments, the recombinant neprosin polypeptide is expressed in a soluble form. In some embodiments, the secreted form is a propolypeptide form of the recombinant neprosin polypeptide.

[0327] In some embodiments, the method is used to express a glycosylated recombinant neprosin polypeptide. In some embodiments, the method is used to express a pre-propeptide of a glycosylated recombinant neprosin polypeptide. In some embodiments, the method is used to express a propeptide of a glycosylated recombinant neprosin polypeptide. In some embodiments, the method is used to express a mature polypeptide of a glycosylated recombinant neprosin polypeptide. In some embodiments, the expressed recombinant neprosin polypeptide is glycosylated at one or more positions 68, 145, 152, or 253, or a combination thereof, wherein the amino acid position is relative to SEQ ID NO: 1. In some embodiments, the expressed recombinant neprosin polypeptide is glycosylated at one or more positions 44, 121, 128, or 229, or a combination thereof, wherein the amino acid position is relative to SEQ ID NO: 8.

[0328] In some embodiments, targeted glycosylation can use a recombinant polynucleotide encoding a recombinant neprosin polypeptide having a mutation at amino acid position 68, 145, 152, or 253, or a combination thereof, wherein the amino acid position is relative to SEQ ID NO: 1, to express a recombinant neprosin polypeptide glycosylated at the specified position. In some embodiments, the expressed recombinant neprosin polypeptide is glycosylated at least at amino acid position 68. In some embodiments, the expressed recombinant neprosin polypeptide is glycosylated at least at amino acid position 145. In some embodiments, the expressed recombinant neprosin polypeptide is glycosylated at least at amino acid position 152. In some embodiments, the expressed recombinant neprosin polypeptide is glycosylated at least at amino acid position 253.

[0329] In some embodiments, the recombinant neprosin polypeptide expressed is non-glycosylated. In some embodiments, the recombinant neprosin expressed is a non-glycosylated pre-propeptide. In some embodiments, the recombinant neprosin expressed is a non-glycosylated propeptide. In some embodiments, the recombinant neprosin expressed is a non-glycosylated mature polypeptide.

[0330] As discussed herein, the method is used to express recombinant neprosin polypeptides, including pre-propolypeptides, propolypeptides or mature forms of recombinant neprosin polypeptides in mammalian cells, insect cells or fungal cells. In some preferred embodiments, the method is used to express recombinant neprosin polypeptides in fungal cells, particularly Pichia pastoris.

[0331] In some embodiments, the expressed recombinant neprosin polypeptide lacks glycosylation at one or more of positions 68, 145, 152, and 253, or a combination thereof, wherein the amino acid position is relative to SEQ ID NO: 1. In some embodiments, the expressed recombinant neprosin polypeptide lacks glycosylation at least at position 68, or the equivalent thereof. In some embodiments, the expressed recombinant neprosin polypeptide lacks glycosylation at least at position 145, or the equivalent thereof. In some embodiments, the expressed recombinant neprosin polypeptide lacks glycosylation at least at position 152, or the equivalent thereof. In some embodiments, the expressed recombinant neprosin polypeptide lacks glycosylation at least at position 253, or the equivalent thereof. In some embodiments, the expressed recombinant neprosin polypeptide lacks glycosylation at least at positions 68 and 152, or the equivalent thereof. In some embodiments, the expressed recombinant neprosin polypeptide lacks glycosylation at least at positions 68 and 253, or the equivalent thereof. In some embodiments, the expressed recombinant neprosin polypeptide lacks glycosylation at least at positions 152 and 253, or their equivalents.

[0332] In some embodiments, the expressed recombinant neprosin polypeptide lacks glycosylation at one or more of positions 44, 121, 128, or 229, or a combination thereof, wherein the amino acid position is relative to SEQ ID NO: 8. In some embodiments, the expressed recombinant neprosin polypeptide lacks glycosylation at least at position 44 or its equivalent. In some embodiments, the expressed recombinant neprosin polypeptide lacks glycosylation at least at position 121 or its equivalent. In some embodiments, the expressed recombinant neprosin polypeptide lacks glycosylation at least at position 128 or its equivalent. In some embodiments, the expressed recombinant neprosin polypeptide lacks glycosylation at least at position 229 or its equivalent. In some embodiments, the expressed recombinant neprosin polypeptide lacks glycosylation at least at positions 44 and 128 or their equivalent. In some embodiments, the expressed recombinant neprosin polypeptide lacks glycosylation at least at positions 44 and 229 or their equivalent. In some embodiments, the expressed recombinant neprosin polypeptide lacks glycosylation at least at positions 128 and 229, or their equivalents. In some embodiments, the expressed recombinant neprosin polypeptide lacks glycosylation at least at positions 121 and 229, or their equivalents.

[0333] In some embodiments, the expressed recombinant neprosin polypeptide comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence corresponding to residues 105 to 356 of SEQ ID NO: 8, or to a reference sequence corresponding to SEQ ID NO: 8, wherein the amino acid sequence comprises a substitution with an amino acid other than asparagine (N) at at least amino acid position 44, 121, 128 or 229, or a combination thereof, wherein the amino acid positions are relative to SEQ ID NO: 8. In some embodiments, the amino acid substitution or amino acid residue at amino acid position 44, 121, 128 or 229, or a combination thereof, is independently selected from A, C, D, E, F, G, H, I, K, L, M, P, Q, R, S, T, V, W, and Y. In some embodiments, the amino acid substitution or amino acid residue at position 44, 121, 128 or 229, or a combination thereof, is A.

[0334] In some embodiments, the expressed recombinant neprosin polypeptide comprises an amino acid sequence comprising residues 105 to 356 of SEQ ID NO: 8, wherein the amino acid sequence comprises a substitution with an amino acid other than asparagine (N) at at least amino acid position 121, 128, or 229, or a combination thereof, wherein the amino acid positions are relative to SEQ ID NO: 8. In some embodiments, the recombinant neprosin polypeptide comprises an amino acid sequence comprising SEQ ID NO: 8, wherein the amino acid sequence comprises a substitution with an amino acid other than asparagine (N) at at least amino acid position 44, 121, 128, or 229, or a combination thereof, wherein the amino acid positions are relative to SEQ ID NO: 8. In some embodiments, the amino acid substitution or amino acid residue at amino acid position 44, 121, 128, or 229, or a combination thereof, is independently selected from A, C, D, E, F, G, H, I, K, L, M, P, Q, R, S, T, V, W, and Y. In some embodiments, the amino acid substitution or amino acid residue at position 44, 121, 128 or 229, or a combination thereof, is A.

[0335] In some embodiments, the expressed recombinant neprosin polypeptide comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence corresponding to residues 105 to 356 of SEQ ID NO: 8, or to a reference sequence corresponding to SEQ ID NO: 8, wherein the amino acid sequence comprises a substitution with an amino acid other than asparagine (N) at at least amino acid position 44, wherein the amino acid position is relative to SEQ ID NO: 8. In some embodiments, the amino acid substitution or amino acid residue at position 44 is selected from A, C, D, E, F, G, H, I, K, L, M, P, Q, R, S, T, V, W, and Y. In some embodiments, the amino acid substitution or amino acid residue at position 44 is A.

[0336] In some embodiments, the expressed recombinant neprosin polypeptide comprises an acid-activated proteolytically active polypeptide derived from a subsequence of or comprising SEQ ID NO: 10. In some embodiments, the expressed recombinant neprosin polypeptide comprises an amino acid sequence comprising residues 105 to 356 of SEQ ID NO: 10. In some embodiments, the expressed recombinant neprosin polypeptide comprises an amino acid sequence comprising SEQ ID NO: 10. In some embodiments, the expressed recombinant neprosin polypeptide comprising residues 105 to 356 of or comprising SEQ ID NO: 10 is glycosylated.

[0337] In some embodiments, the expressed recombinant neprosin polypeptide comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence corresponding to residues 105 to 356 of SEQ ID NO: 8, or to a reference sequence corresponding to SEQ ID NO: 8, wherein the amino acid sequence comprises a substitution with an amino acid other than asparagine (N) at at least amino acid position 121, wherein the amino acid position is relative to SEQ ID NO: 8. In some embodiments, the amino acid substitution or amino acid residue at position 121 is selected from A, C, D, E, F, G, H, I, K, L, M, P, Q, R, S, T, V, W, and Y. In some embodiments, the amino acid substitution or amino acid residue at position 121 is A.

[0338] In some embodiments, the expressed recombinant neprosin polypeptide comprises an acid-activated proteolytically active polypeptide derived from a subsequence of or comprising SEQ ID NO: 12. In some embodiments, the expressed recombinant neprosin polypeptide comprises an amino acid sequence comprising residues 105 to 356 of SEQ ID NO: 12. In some embodiments, the expressed recombinant neprosin polypeptide comprises an amino acid sequence comprising SEQ ID NO: 12. In some embodiments, the expressed recombinant neprosin polypeptide comprising residues 105 to 356 of or comprising SEQ ID NO: 12 is glycosylated.

[0339] In some embodiments, the expressed recombinant neprosin polypeptide comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence corresponding to residues 105 to 356 of SEQ ID NO: 8, or to a reference sequence corresponding to SEQ ID NO: 8, wherein the amino acid sequence comprises a substitution with an amino acid other than asparagine (N) at at least amino acid position 128, wherein the amino acid position is relative to SEQ ID NO: 8. In some embodiments, the amino acid substitution or amino acid residue at position 128 is selected from A, C, D, E, F, G, H, I, K, L, M, P, Q, R, S, T, V, W, and Y. In some embodiments, the amino acid substitution or amino acid residue at position 128 is A.

[0340] In some embodiments, the expressed recombinant neprosin polypeptide comprises an acid-activated proteolytically active polypeptide derived from a subsequence of or comprising SEQ ID NO: 14. In some embodiments, the expressed recombinant neprosin polypeptide comprises an amino acid sequence comprising residues 105 to 356 of SEQ ID NO: 14. In some embodiments, the expressed recombinant neprosin polypeptide comprises an amino acid sequence comprising SEQ ID NO: 14. In some embodiments, the expressed recombinant neprosin polypeptide comprising residues 105 to 356 of or comprising SEQ ID NO: 14 is glycosylated.

[0341] In some embodiments, the expressed recombinant neprosin polypeptide comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a sequence corresponding to residues 105 to 356 of SEQ ID NO: 8, or to a sequence corresponding to SEQ ID NO: 8, wherein the amino acid sequence comprises a substitution with an amino acid other than asparagine (N) at least at amino acid position 229, or the equivalent thereof, wherein the amino acid position is relative to SEQ ID NO: 8. In some embodiments, the amino acid substitution or amino acid residue at position 229 is selected from A, C, D, E, F, G, H, I, K, L, M, P, Q, R, S, T, V, W, and Y. In some embodiments, the amino acid substitution or amino acid residue at position 229 is A.

[0342] In some embodiments, the expressed recombinant neprosin polypeptide comprises an acid-activated proteolytically active polypeptide derived from a subsequence of or comprising SEQ ID NO: 16. In some embodiments, the expressed recombinant neprosin polypeptide comprises an amino acid sequence comprising residues 105 to 356 of SEQ ID NO: 16. In some embodiments, the expressed recombinant neprosin polypeptide comprises an amino acid sequence comprising SEQ ID NO: 16. In some embodiments, the recombinant neprosin polypeptide comprising residues 105 to 356 of SEQ ID NO: 12 or an amino acid sequence comprising SEQ ID NO: 16 is glycosylated.

[0343] In some embodiments, the expressed recombinant neprosin polypeptide comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence corresponding to residues 105 to 356 of SEQ ID NO: 8, or to a reference sequence corresponding to SEQ ID NO: 8, wherein the amino acid sequence comprises a substitution with an amino acid other than asparagine (N) at at least amino acid positions 121 and 229, wherein the amino acid positions are relative to SEQ ID NO: 8. In some embodiments, the amino acid substitution or amino acid residue at each of positions 121 and 229 is independently selected from A, C, D, E, F, G, H, I, K, L, M, P, Q, R, S, T, V, W, and Y. In some embodiments, the amino acid substitution or amino acid residue at positions 121 and 229 is A.

[0344] In some embodiments, the expressed recombinant neprosin polypeptide comprises an acid-activated proteolytically active polypeptide derived from a subsequence of or comprising SEQ ID NO: 18. In some embodiments, the expressed recombinant neprosin polypeptide comprises an amino acid sequence comprising residues 105 to 356 of SEQ ID NO: 18. In some embodiments, the expressed recombinant neprosin polypeptide comprises an amino acid sequence comprising SEQ ID NO: 18. In some embodiments, the recombinant neprosin polypeptide comprising residues 105 to 356 of or comprising SEQ ID NO: 18 is glycosylated.

[0345] In some embodiments, the expressed recombinant neprosin polypeptide comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence corresponding to residues 105 to 356 of SEQ ID NO: 8, or to a reference sequence corresponding to SEQ ID NO: 8, wherein the amino acid sequence comprises a substitution with an amino acid other than asparagine (N) at at least amino acid positions 44, 128, and 229, wherein the amino acid positions are relative to SEQ ID NO: 8. In some embodiments, the amino acid substitution or amino acid residue at each of positions 44, 128, and 229 is independently selected from A, C, D, E, F, G, H, I, K, L, M, P, Q, R, S, T, V, W, and Y. In some embodiments, the amino acid substitution or amino acid residue at positions 44, 128, and 229 is A.

[0346] In some embodiments, the expressed recombinant neprosin polypeptide comprises an acid-activated proteolytically active polypeptide derived from a subsequence of or comprising SEQ ID NO: 20. In some embodiments, the expressed recombinant neprosin polypeptide comprises an amino acid sequence comprising residues 105 to 356 of SEQ ID NO: 20. In some embodiments, the recombinant neprosin polypeptide comprises an amino acid sequence comprising SEQ ID NO: 20. In some embodiments, the recombinant neprosin polypeptide comprising residues 105 to 356 of or comprising SEQ ID NO: 20 is glycosylated.

[0347] In some embodiments, the expressed recombinant neprosin polypeptide comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence corresponding to residues 105 to 356 of SEQ ID NO: 8, or to a reference sequence corresponding to SEQ ID NO: 8, wherein the amino acid sequence comprises a substitution with an amino acid other than asparagine (N) at at least amino acid positions 44, 121, 128, and 229, wherein the amino acid positions are relative to SEQ ID NO: 8. In some embodiments, the amino acid substitution or amino acid residue at each of positions 44, 121, 128, and 229 is independently selected from A, C, D, E, F, G, H, I, K, L, M, P, Q, R, S, T, V, W, and Y. In some embodiments, the amino acid substitution or amino acid residue at positions 44, 121, 128, and 229 is A.

[0348] In some embodiments, the expressed recombinant neprosin polypeptide comprises an acid-activated proteolytically active polypeptide derived from a subsequence of SEQ ID NO: 22 or comprising a subsequence of SEQ ID NO: 22. In some embodiments, the expressed recombinant neprosin polypeptide comprises an amino acid sequence comprising residues 105 to 356 of SEQ ID NO: 22. In some embodiments, the expressed recombinant neprosin polypeptide comprises an amino acid sequence comprising SEQ ID NO: 22. In some embodiments, the expressed recombinant neprosin polypeptide comprising residues 105 to 356 of SEQ ID NO: 22 or comprising an amino acid sequence of SEQ ID NO: 22 is not glycosylated.

[0349] In some embodiments, the expressed recombinant neprosin polypeptide comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence corresponding to residues 105 to 356 of SEQ ID NO: 8, or to a reference sequence corresponding to SEQ ID NO: 8, wherein the amino acid sequence comprises a substitution with an amino acid other than asparagine (N) at at least amino acid positions 44 and 229, wherein the amino acid positions are relative to SEQ ID NO: 8. In some embodiments, the amino acid substitution or amino acid residue at each of positions 44 and 229 is independently selected from A, C, D, E, F, G, H, I, K, L, M, P, Q, R, S, T, V, W, and Y. In some embodiments, the amino acid substitution or amino acid residue at positions 44 and 229 is A.

[0350] In some embodiments, the expressed recombinant neprosin polypeptide comprises an acid-activated proteolytically active polypeptide derived from a subsequence of or comprising SEQ ID NO: 32. In some embodiments, the expressed recombinant neprosin polypeptide comprises an amino acid sequence comprising residues 105 to 356 of SEQ ID NO: 32. In some embodiments, the expressed recombinant neprosin polypeptide comprises an amino acid sequence comprising SEQ ID NO: 32. In some embodiments, the expressed recombinant neprosin polypeptide comprising residues 105 to 356 of or comprising SEQ ID NO: 32 is glycosylated.

[0351] In some embodiments, the expressed recombinant neprosin polypeptide comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the sequence corresponding to residues 105 to 356 of SEQ ID NO: 8, or to the sequence corresponding to SEQ ID NO: 8.

[0352] In some embodiments, the expressed recombinant neprosin polypeptide comprises an acid-activated proteolytically active polypeptide derived from a subsequence of SEQ ID NO: 8 or comprising a subsequence of SEQ ID NO: 8. In some embodiments, the expressed recombinant neprosin polypeptide comprises an amino acid sequence comprising residues 105 to 356 of SEQ ID NO: 8. In some embodiments, the expressed recombinant neprosin polypeptide comprises an amino acid sequence comprising SEQ ID NO: 8. In some embodiments, the expressed recombinant neprosin polypeptide comprising amino acid sequence residues 105 to 356 of SEQ ID NO: 8 or comprising SEQ ID NO: 22 is glycosylated.

[0353] In some embodiments, the expressed recombinant neprosin polypeptide comprises an amino acid sequence comprising residues 26 to 381 of SEQ ID NO: 44, 46, 48, 50, or 52. In some embodiments, the expressed recombinant neprosin polypeptide comprises a propolypeptide comprised within residues 26 to 381 of SEQ ID NO: 44, 46, 48, 50, or 52. In some embodiments, the expressed recombinant neprosin polypeptide comprises a mature neprosin polypeptide comprised within residues 26 to 381 of SEQ ID NO: 44, 46, 48, 50, or 52. In some embodiments, the expressed recombinant neprosin polypeptide comprises an amino acid sequence comprising SEQ ID NO: 36, 38, 40, 42, 44, 46, 48, 50, or 52.

[0354] In some embodiments, the expressed recombinant neprosin polypeptide comprises an amino acid sequence comprising residues 96 to 451 of SEQ ID NO: 58, 60, 62, 64, 66, 68, or 70. In some embodiments, the expressed recombinant neprosin polypeptide comprises a propolypeptide comprised within residues 96 to 451 of SEQ ID NO: 58, 60, 62, 64, 66, 68, or 70. In some embodiments, the expressed recombinant neprosin polypeptide comprises a mature neprosin polypeptide comprised within residues 96 to 451 of SEQ ID NO: 58, 60, 62, 64, 66, 68, or 70. In some embodiments, the expressed recombinant neprosin polypeptide comprises an amino acid sequence comprising SEQ ID NO: 54, 56, 58, 60, 62, 64, 66, 68, or 70.

[0355] In some embodiments, the method of preparing a mature neprosin polypeptide comprises treating a full-length neprosin polypeptide or a neprosin pre-propolypeptide or pro-polypeptide prepared from expressed neprosin described herein to an acidic pH; or treating a full-length neprosin polypeptide or a neprosin pre-propolypeptide or pro-polypeptide of a recombinant neprosin variant described herein to an acidic pH under suitable conditions for forming a mature neprosin polypeptide. In some embodiments, the treatment is performed at a pH of about 2 to about 6. In some embodiments, the treatment is performed at a pH of about 2 to about 4.5. In some embodiments, the treatment is performed at a pH of about 2 to about 3. In some embodiments, the method of preparing a mature neprosin polypeptide is performed in vitro. In some embodiments, the mature neprosin polypeptide produced by the method has a molecular weight of about 25.5 kDa to about 29 kDa, particularly as determined by SDS-PAGE.

[0356] In some embodiments, the propeptide is removed at an acidic pH, thereby activating the enzyme and forming a mature neprosin polypeptide. In some embodiments, the propeptide is cleaved by a cellular protease, thereby activating the enzyme and forming a mature neprosin polypeptide. In some embodiments, the cell expresses a mature form of a neprosin polypeptide.

[0357] In some embodiments, the present disclosure provides mature, proteolytically active recombinant neprosin polypeptides prepared by acid activation of a prepropolypeptide or propolypeptide of a recombinant neprosin polypeptide described herein. Medicinal and dietary / nutritional compositions

[0358] In other aspects, recombinant neprosin is formulated as a composition. In some embodiments, the composition comprises a recombinant pre-propolypeptide or pre-proenzyme form of a neprosin polypeptide as described herein, a pro-polypeptide or pro-enzyme form of a neprosin polypeptide, or a mature neprosin polypeptide. In some embodiments, the recombinant neprosin polypeptide in the composition is prepared from an expressed recombinant neprosin polypeptide or a recombinant neprosin polypeptide variant, including a pre-propolypeptide, pro-polypeptide or mature polypeptide form of a variant as described herein. In some embodiments, the mature neprosin polypeptide in the composition has a molecular weight of about 25.5 kDa to about 29 kDa, for example, as determined by SDS-PAGE. In some embodiments, the recombinant neprosin polypeptide in the composition is prepared from a recombinant neprosin polypeptide expressed or produced in a mammalian cell, an insect cell, or a fungal cell.

[0359] In some embodiments, the recombinant neprosin is formulated as a pharmaceutical composition, a dietary / nutritional supplement, or a food composition.

[0360] In some embodiments, the recombinant neprosin polypeptide can be used in any suitable edible enzyme delivery matrix. In some embodiments, the recombinant neprosin polypeptide is present in an edible enzyme delivery matrix designed to allow rapid dispersion of the polypeptide in the digestive tract after ingestion of neprosin by an animal or subject.

[0361] In some embodiments, recombinant neprosin is formulated into a pharmaceutical or dietary composition. Depending on the mode of administration, the composition comprises a therapeutically effective amount of a recombinant neprosin polypeptide and may be in the form of a solid, semisolid or liquid. The term "pharmaceutically acceptable" means approved by a regulatory agency of the federal or state government or listed in the U.S. Pharmacopoeia or other generally recognized pharmacopeias for use in animals and more particularly in humans. The term "carrier" refers to a diluent, adjuvant, excipient or vehicle administered with a therapeutic agent. Such a pharmaceutical carrier may be a sterile liquid, such as water and oil, including those of petroleum, animal, plant or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, etc. Saline solutions and aqueous solutions of glucose and glycerol may also be used as liquid carriers.

[0362] In some embodiments, the present disclosure provides a pharmaceutical composition comprising a recombinant neprosin polypeptide and a pharmaceutically acceptable carrier, excipient or diluent. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glyceryl monostearate, talc, sodium chloride, skim milk powder, glycerol, propylene glycol, water, ethanol and the like. If desired, the composition may also include a small amount of a wetting agent or emulsifier, or a pH buffer. These compositions may take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained release formulations, etc. Examples of suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences" by EW Martin, which is incorporated herein by reference in its entirety. Such a composition will contain a therapeutically effective amount of the enzyme, preferably in a purified form, and an appropriate amount of carrier, so as to provide a form for appropriate administration to a subject. The formulation should be suitable for the mode of administration.

[0363] In some embodiments, the recombinant neprosin polypeptide is formulated for use as an oral pharmaceutical composition (e.g., for oral administration). Any suitable form for use in delivering the neprosin polypeptide can be used, including but not limited to pills, tablets, gel tablets, capsules, lozenges, dragees, powders, soft gels, sol-gels, gels, emulsions, implants, patches, sprays, ointments, liniments, creams, pastes, gels, paints, aerosols, chewing gums, demulcents, sticks, suspensions (including but not limited to oil-based suspensions, oil-in-water emulsions, etc.), slurries, syrups, controlled release formulations, suppositories, etc. For oral administration, the pharmaceutical composition can be used alone or in combination with appropriate additives to prepare tablets, powders, granules, capsules, syrups, liquids, suspensions, etc. For example, the solid oral form of the composition can be prepared with conventional additives, disintegrants, lubricants, diluents, buffers, wetting agents, preservatives, and flavoring agents. Non-limiting examples of excipients include sugars (e.g., lactose, sucrose, mannitol and / or sorbitol), starches (e.g., corn starch, wheat starch, rice starch, potato starch or other plant starches), celluloses (e.g., methylcellulose, hydroxypropylmethylcellulose, sodium carboxymethylcellulose), gums (e.g., gum arabic, tragacanth, guar gum, etc.), and / or proteins (e.g., gelatin, collagen, etc.). Additional components in oral formulations may include colorants and or sweeteners (e.g., glucose, sucrose, and mannitol) and lubricants (e.g., magnesium stearate), as well as enteric coatings (e.g., methacrylate polymers, hydroxypropylmethylcellulose phthalate, and / or any other suitable enteric coating known in the art). In some embodiments, the formulation releases enzymes in the stomach of the subject so that the peptide food antigens can be enzymatically degraded.

[0364] In some embodiments, the recombinant neprosin polypeptide is provided as a unit dose formulation. For example, but not limited to, the unit dose can be present in tablets, capsules, etc. The unit dose can be solid, liquid, powder or any other form. The unit dose formulation of the pharmaceutical composition will allow for appropriate administration (e.g., based on the amount of immunogenic protein ingested) while avoiding the potential negative side effects of administering an excessive amount of the composition.

[0365] In some embodiments, the recombinant neprosin polypeptide or a composition thereof (including as a pharmaceutical composition) can be lyophilized from an aqueous solution, optionally in the presence of an appropriate buffer (e.g., phosphate, citrate, histidine, imidazole buffer) and an excipient (e.g., a cryoprotectant such as sucrose, lactose, trehalose). The lyophilized cake can be optionally blended with an excipient and prepared into different forms.

[0366] Without being bound by theory, it is believed that neprosin may be less active or substantially inactive at neutral to alkaline pH. This may be important in the presence of the possibility of undesirable digestion by enzymes. For example, in the case of oral administration of pharmaceutical compositions, buffering the composition to pH 6.5 or higher may result in a less active form of the enzyme, so that oral mucosa, esophageal mucosa and other cells that may contact the composition will not be digested by the enzymes therein. Similarly, when the composition is added to food, the buffered enzyme will not (or not quite) digest the food before the food is eaten. In such a case, introducing the composition into the acidic environment of the stomach may result in pH reduction and enzyme activation.

[0367] Therefore, in some embodiments, the pharmaceutical composition is buffered to about pH 6.5 or higher. In preferred embodiments, the composition is buffered to about pH 6.5 to about pH 8.5. In some embodiments, the composition is in liquid form. In some embodiments, the composition is in semisolid form. In some embodiments, the composition is in solid form. In some embodiments, the pH of the composition is adjusted in liquid form, and the composition is dried to form a solid. In some embodiments, the enzyme in the composition is activated when in contact with acid (i.e., in the stomach).

[0368] In some embodiments, a pharmaceutical composition comprising a recombinant neprosin polypeptide is formulated in a delayed release vehicle such that the enzyme is continuously released while the formulation is present in the stomach. In some embodiments, the formulation has a pH greater than about 5 prior to contact with the acid in the stomach. In some embodiments, the formulation comprises a biologically acceptable buffer such that the pH of the composition is maintained at about pH 5 or 6 for at least a period of time after contact with the acid in the stomach. In preferred embodiments, the formulation is a controlled release formulation. The term "controlled release formulation" includes sustained release and time release formulations. Controlled release formulations are well known in the art. These include excipients that allow for sustained, periodic, pulsatile or delayed release of the drug. Controlled release formulations include, but are not limited to, embedding the drug into a matrix; enteric coatings; microencapsulation; gels and hydrogels; and any other formulation that allows controlled release of the drug.

[0369] It should also be understood that in some embodiments, proteolytically active neprosin polypeptides can be used in compositions, such as proteolytically active mature neprosin polypeptides. In some embodiments, the proteolytically active mature neprosin polypeptide in the composition is treated to an acidic pH to prepare an active mature polypeptide. In some embodiments, the proteolytically active neprosin polypeptide can be used to treat food prior to ingestion or be orally administered simultaneously with food ingestion.

[0370] In some embodiments, the composition is a food comprising a recombinant neprosin polypeptide. In some embodiments, the food is a liquid, semisolid or solid. In some embodiments, the food comprises gluten or is suspected of comprising gluten, such as baked products made from wheat, rye and barley (e.g., cakes, muffins, donuts, pastries, rolls and bread), pasta, cookies, corn flakes, cereals, etc. In some embodiments, the food can be eaten with another food comprising gluten or suspected of comprising gluten. Non-limiting examples of such food include powders, spreads, sprays, sauces, dips, whipped cream, candy, chewing gum, syrups, sugar, salt, salad dressings, spices, cheese, butter, margarine, spreads, butter, frying shortening, mayonnaise, dairy products, nut butter, seed butter, nut butter, peanut butter, etc.

[0371] In some embodiments, the recombinant neprosin polypeptide or a composition thereof is mixed with food, or used to pretreat food containing gluten. The recombinant neprosin polypeptide in the food may have enzymatic activity to reduce the level of gluten in the food before or during ingestion. In some embodiments, the recombinant neprosin polypeptide is dispersed into the food before consumption, optionally at a pH at which it is inactive, such as a pH of about or above 5. Preferably, the food or additive containing the neprosin polypeptide does not need to be heated before being ingested by the patient, so that the loss of enzyme activity that may result from the elevated temperature can be minimized.

[0372] In some embodiments, a dispenser comprising an inert excipient and an effective amount of a recombinant neprosin polypeptide or a composition thereof is used to dispense neprosin or a composition thereof to digest gluten in food. In some embodiments, the recombinant neprosin polypeptide or a composition thereof and / or an inert excipient is added to the food before the food is consumed. In some embodiments, the inert excipient comprises sodium chloride or sodium iodide or a mixture thereof. In some embodiments, the pharmaceutical composition and / or the inert excipient is in the form of particles, the size of which is designed to be effectively dispensed from the dispenser.

[0373] In some embodiments, the recombinant neprosin polypeptide or a composition thereof (e.g., such as a pharmaceutical composition, an edible composition, or a composition with an additional protease) or a food product comprises from about 0.1% to about 99%, from about 0.5% to about 95%, from about 1% to about 95%, from about 5% to about 95%, from about 10% to about 90%, from about 20% to about 80%, from about 25% to about 75% neprosin polypeptide. In some embodiments, the amount of the recombinant neprosin polypeptide in the composition (such as a pharmaceutical composition or an edible composition) or food product is about 0.01%, about 0.1%, about 0.5%, about 1%, about 5%, about 10%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, or about 95% of the total composition or food product, or a range (including endpoints) between any two of these values.

[0374] In some embodiments, the composition comprising the pharmaceutical composition comprises a recombinant neprosin polypeptide and one or more additional proteases. In some embodiments, one or more additional proteases are aspartic proteases, serine proteases, threonine proteases, cysteine ​​proteases, glutamic proteases or metalloproteases. In some embodiments, as described herein, the additional proteases are Nepenthes proteases, such as Nepenthes proteases I and / or Nepenthes proteases II. In some embodiments, the composition comprises one or more additional exoproteases, such as leucine aminopeptidases and carboxypeptidases. In some embodiments, one or more additional proteases are trypsin. In preferred embodiments, one or more additional proteases are active at acidic pH (e.g., pH 2-6). Exemplary proteases that can be used in the present invention include, but are not limited to, Nepenthes proteases I, Nepenthes proteases II, BACE, cathepsin D, cathepsin E, chymosin (or "renin"), napsin, pepsin, plasmepsin, presenilin, renin, trypsin, chymotrypsin, elastase and cysteine ​​endoproteinase (EP) B2 (also known as EPB2). In a preferred embodiment, the at least one additional protease is active at acidic pH, such as the pH found in the stomach (eg, pH 1.5 to 3.5). Combination with Nepenthes protease

[0375] In some embodiments, the composition comprising a recombinant neprosin polypeptide comprises an additional protease, the additional protease being a Nepenthes protease, such as one or more of Nepenthes protease I and Nepenthes protease II. Nepenthes proteases (EC 3.4.23.12) are a class of aspartic proteases found in Nepenthes secretions and a variety of other plant sources (see, e.g., Tokes et al., Digestive Enzymes Secreted by the Carnivorous Plant Nepenthes macferlanei L., Planta (Berl.), 1974, 119:39-46). Nepenthes proteases have two known isoforms: Nepenthes protease I (known to have two variants: Nepenthes protease Ia and Nepenthes protease Ib) and Nepenthes protease II. Nepenthes protease I mRNA / cDNA sequences and corresponding amino acid sequences have been described from several Nepenthes species, including Nepenthes mirabilis (GenBank Accession Nos. JX494401 and AFV26024); Nepenthes gracilis (GenBank Accession Nos. AB114914 and BAD07474) and Nepenthes alata (GenBank Accession Nos. AB266803 and BAF98915), among others. Nepenthes protease II mRNA / cDNA sequences have been described from several Nepenthes species, including Nepenthes mirabilis (GenBank Accession Nos. JX494402 and AFV26025) and Nepenthes gracilis (GenBank Accession Nos. AB114915 and BAD07475.1), among others. These sequences are also found in U.S. Patent Application Publication No. 20140186330, which is incorporated herein by reference in its entirety. Each sequence represented by a GenBank accession number provided herein is incorporated herein by reference in its entirety. Exemplary Nepenthes protease I and Nepenthes protease II proteases are presented as SEQ ID NOs: 72, 73, 74, 75, 76, and 77. In some embodiments, biosynthesis of Nepenthes proteases can be achieved by transforming cells with a vector comprising a cDNA encoding a Nepenthes protease.

[0376] In some embodiments, the Nepenthes protease is a recombinant Nepenthes protease having at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence of Nepenthes protease I. In some embodiments, the Nepenthes protease comprises the amino acid sequence of Nepenthes protease I.

[0377] In some embodiments, the Nepenthes protease is a variant of a Nepenthes protease having at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence of Nepenthes protease II. In some embodiments, the Nepenthes protease comprises the amino acid sequence of Nepenthes protease II.

[0378] In some embodiments, Nepenthes protease I, Nepenthes protease II or its variants are synthesized by transfecting, infecting or transforming cells with one or more vectors containing the cDNA sequence of each desired enzyme. In some embodiments, a single cell, cell line or organism can be engineered to produce two or more enzymes. In some embodiments, the desired enzyme is synthesized by a separate cell and combined in a pharmaceutical composition. In a preferred embodiment, recombinant Nepenthes protease I, Nepenthes protease II or its variants are not glycosylated. In some embodiments, recombinant Nepenthes protease I, Nepenthes protease II or its variants have a glycosylation pattern different from that of the natural enzyme (i.e., Nepenthes protease I or Nepenthes protease II, separated from Nepenthes plants). Synthetic (e.g., recombinant) Nepenthes enzymes can be concentrated or purified according to known methods, such as those methods for separating Nepenthes enzymes from plant Nepenthes liquids.

[0379] In some embodiments, the composition comprises neprosin and Nepenthes proteases or variants thereof. In some embodiments, the composition comprises neprosin and Nepenthes proteases I or variants thereof. In some embodiments, the composition comprises neprosin and Nepenthes proteases II or variants thereof. In some embodiments, the composition comprises neprosin, Nepenthes proteases I and Nepenthes proteases II or variants thereof. In some embodiments, the composition comprises a ratio of neprosin to Nepenthes proteases (or their respective variants) of at least about 100:1. In some embodiments, the composition comprises a ratio of neprosin to Nepenthes proteases of at least about 90:1. In some embodiments, the composition comprises a ratio of neprosin to Nepenthes proteases of at least about 70:1. In some embodiments, the composition comprises a ratio of neprosin to Nepenthes proteases of at least about 60:1. In some embodiments, the composition comprises a ratio of neprosin to Nepenthes proteases of at least about 50:1. In some embodiments, the composition comprises a ratio of neprosin to Nepenthes proteases of at least about 40:1. In some embodiments, the composition comprises a ratio of neprosin to Nepenthes proteases of at least about 30:1. In some embodiments, the composition comprises a ratio of neprosin to Nepenthes proteases of at least about 20:1. In some embodiments, the composition comprises a ratio of neprosin to Nepenthes proteases of at least about 10:1. In some embodiments, the composition comprises a ratio of neprosin to Nepenthes proteases of at least about 5:1. In some embodiments, the composition comprises a ratio of neprosin to Nepenthes proteases of at least about 4:1. In some embodiments, the composition comprises a ratio of neprosin to Nepenthes proteases of at least about 3:1. In some embodiments, the composition comprises a ratio of neprosin to Nepenthes proteases of at least about 2:1. In some embodiments, the composition comprises a ratio of neprosin to Nepenthes proteases of at least about 1:1. In some embodiments, the composition comprises a ratio of neprosin to Nepenthes proteases of at least about 1:2. In some embodiments, the composition comprises a ratio of neprosin to Nepenthes proteases of at least about 1:3. In some embodiments, the composition comprises a ratio of neprosin to Nepenthes proteases of at least about 1:4. In some embodiments, the composition comprises a ratio of neprosin to Nepenthes proteases of at least about 1:5. In some embodiments, the composition comprises a ratio of neprosin to Nepenthes proteases of at least about 1:10. In some embodiments, the composition comprises a ratio of neprosin to Nepenthes proteases of at least about 1:20.In some embodiments, the composition comprises a ratio of neprosin to Nepenthes proteases of at least about 1:30. In some embodiments, the composition comprises a ratio of neprosin to Nepenthes proteases of at least about 1:40. In some embodiments, the composition comprises a ratio of neprosin to Nepenthes proteases of at least about 1:50. In some embodiments, the composition comprises a ratio of neprosin to Nepenthes proteases of at least about 1:60. In some embodiments, the composition comprises a ratio of neprosin to Nepenthes proteases of at least about 1:70. In some embodiments, the composition comprises a ratio of neprosin to Nepenthes proteases of at least about 1:80. In some embodiments, the composition comprises a ratio of neprosin to Nepenthes proteases of at least about 1:90. In some embodiments, the composition comprises a ratio of neprosin to Nepenthes proteases of at least about 1:100.

[0380] In some embodiments, the composition comprises neprosin and substantially only recombinant Nepenthes protease I or a variant thereof. In some embodiments, the composition comprises neprosin and substantially only Nepenthes protease II or a variant thereof. In some embodiments, the composition comprises neprosin and Nepenthes protease I and Nepenthes protease II or a variant thereof. In some embodiments, the composition comprises neprosin and a recombinant Nepenthes protease or a variant thereof in a ratio of at least about 100:1 of Nepenthes protease I to Nepenthes protease II (or their respective variants). In some embodiments, the composition comprises neprosin and a recombinant Nepenthes protease or a variant thereof in a ratio of at least about 90:1 of Nepenthes protease I to Nepenthes protease II. In some embodiments, the composition comprises neprosin and a recombinant Nepenthes protease or a variant thereof in a ratio of at least about 70:1 of Nepenthes protease I to Nepenthes protease II. In some embodiments, the composition comprises neprosin and a recombinant Nepenthes protease or a variant thereof in a ratio of at least about 60:1 of Nepenthes protease I to Nepenthes protease II. In some embodiments, the composition comprises neprosin and a recombinant Nepenthes protease or variant thereof in a ratio of Nepenthes protease I to Nepenthes protease II of at least about 50:1. In some embodiments, the composition comprises neprosin and a recombinant Nepenthes protease or variant thereof in a ratio of Nepenthes protease I to Nepenthes protease II of at least about 40:1. In some embodiments, the composition comprises neprosin and a recombinant Nepenthes protease or variant thereof in a ratio of Nepenthes protease I to Nepenthes protease II of at least about 30:1. In some embodiments, the composition comprises neprosin and a recombinant Nepenthes protease or variant thereof in a ratio of Nepenthes protease I to Nepenthes protease II of at least about 20:1. In some embodiments, the composition comprises neprosin and a recombinant Nepenthes protease or variant thereof in a ratio of Nepenthes protease I to Nepenthes protease II of at least about 10:1. In some embodiments, the composition comprises neprosin and a recombinant Nepenthes protease or variant thereof in a ratio of Nepenthes protease I to Nepenthes protease II of at least about 5:1. In some embodiments, the composition comprises neprosin and a recombinant Nepenthes protease or variant thereof in a ratio of Nepenthes protease I to Nepenthes protease II of at least about 4: 1. In some embodiments, the composition comprises neprosin and a recombinant Nepenthes protease or variant thereof in a ratio of Nepenthes protease I to Nepenthes protease II of at least about 3: 1. In some embodiments, the composition comprises neprosin and a recombinant Nepenthes protease or variant thereof in a ratio of Nepenthes protease I to Nepenthes protease II of at least about 2: 1.In some embodiments, the composition comprises neprosin and neprosin and a recombinant Nepenthes protease or variant thereof, wherein the ratio of Nepenthes protease I to Nepenthes protease II is at least about 1:1. In some embodiments, the composition comprises neprosin and a recombinant Nepenthes protease or variant thereof in a ratio of Nepenthes protease I to Nepenthes protease II of at least about 1:2. In some embodiments, the composition comprises neprosin and a recombinant Nepenthes protease or variant thereof in a ratio of Nepenthes protease I to Nepenthes protease II of at least about 1:3. In some embodiments, the composition comprises neprosin and a recombinant Nepenthes protease or variant thereof in a ratio of Nepenthes protease I to Nepenthes protease II of at least about 1:4. In some embodiments, the composition comprises neprosin and a recombinant Nepenthes protease or variant thereof in a ratio of Nepenthes protease I to Nepenthes protease II of at least about 1:5. In some embodiments, the composition comprises neprosin and a recombinant Nepenthes protease or variant thereof in a ratio of Nepenthes protease I to Nepenthes protease II of at least about 1:10. In some embodiments, the composition comprises neprosin and a recombinant Nepenthes protease or variant thereof in a ratio of Nepenthes protease I to Nepenthes protease II of at least about 1:20. In some embodiments, the composition comprises neprosin and a recombinant Nepenthes protease or variant thereof in a ratio of Nepenthes protease I to Nepenthes protease II of at least about 1:30. In some embodiments, the composition comprises neprosin and a recombinant Nepenthes protease or variant thereof in a ratio of Nepenthes protease I to Nepenthes protease II of at least about 1:40. In some embodiments, the composition comprises neprosin and a recombinant Nepenthes protease or variant thereof in a ratio of Nepenthes protease I to Nepenthes protease II of at least about 1:50. In some embodiments, the composition comprises neprosin and a recombinant Nepenthes protease or variant thereof in a ratio of Nepenthes protease I to Nepenthes protease II of at least about 1:60. In some embodiments, the composition comprises neprosin and a recombinant Nepenthes protease or variant thereof in a ratio of Nepenthes protease I to Nepenthes protease II of at least about 1:70. In some embodiments, the composition comprises neprosin and a recombinant Nepenthes protease or variant thereof in a ratio of Nepenthes protease I to Nepenthes protease II of at least about 1:80. In some embodiments, the composition comprises neprosin and a recombinant Nepenthes protease or variant thereof in a ratio of Nepenthes protease I to Nepenthes protease II of at least about 1:90. In some embodiments, the composition comprises neprosin and a recombinant Nepenthes protease or variant thereof in a ratio of Nepenthes protease I to Nepenthes protease II of at least about 1:100.

[0381] In some embodiments, the ratio of neprosin to Nepenthes proteases I and / or II in the composition is such that the peptide food antigen is cleaved into sufficiently small and / or harmless fragments to prevent, reduce or attenuate gluten intolerance, celiac disease, wheat allergy or dermatitis herpetiformis, inflammation, IEL proliferation or recruitment, intraepithelial lymphocytosis and / or villous atrophy or any symptoms thereof in the intestinal tract of a subject. In some embodiments, the ratio of neprosin: Nepenthes proteases is between about 1:100 to about 100:1.

[0382] In some embodiments, the Nepenthes protease I or Nepenthes protease II in the composition is a full-length Nepenthes protease I or Nepenthes protease II. In some embodiments, the Nepenthes protease I or Nepenthes protease II in the composition is a mature form of Nepenthes protease I or Nepenthes protease II. In some embodiments, the Nepenthes protease I or Nepenthes protease II in the composition is a zymogen of Nepenthes protease I or Nepenthes protease II.

[0383] Regarding the zymogen or propolypeptide of Nepenthes proteases, it is known that Nepenthes proteases I and / or Nepenthes proteases II exist as zymogens in Nepenthes, and the propolypeptide or prozymogen is cleaved to activate the enzyme in the Nepenthes fluid. In some embodiments, the composition comprises a recombinant neprosin in the form described above and a zymogen / propeptide of Nepenthes proteases I and / or Nepenthes proteases II. In some embodiments, the propeptide is adjacent to the N-terminus of the enzyme. In some embodiments, the propeptide is a naturally occurring propeptide of the enzyme. In some embodiments, the propeptide is a heterologous propeptide (e.g., from a different protein or species, or synthetic). In some embodiments, the propeptide is cleaved by acidic conditions. In some embodiments, the propeptide is cleaved by the enzyme. In some embodiments, the presence of the propeptide results in a delay in the activity of the enzyme in the stomach (e.g., due to the time required to remove the propeptide and produce the mature enzyme). In some embodiments, the propeptide is engineered to be removed more slowly to delay the activity of the enzyme in the stomach. In some embodiments, the propeptide is engineered to be removed faster to accelerate the activity of the enzyme in the stomach. Uses and methods

[0384] The recombinant neprosin polypeptides described herein, alone or as a composition with other proteases, can be used to degrade dietary proteins, particularly proteins rich in proline and / or glutamine or other immunogenic proteins that cannot be effectively degraded by digestive tract enzymes. Such degradation of proteins will increase protein absorption and / or reduce immunogenicity, which may have a beneficial effect on the symptoms of intestinal diseases and disorders, such as celiac disease, gluten intolerance, irritable bowel syndrome, colitis, Crohn's disease, food allergies, etc. Therefore, in another aspect, the present disclosure provides a method for regulating a condition mediated by gluten intolerance in a subject, the method comprising administering an effective amount of a recombinant neprosin polypeptide described herein to a subject in need thereof. In some embodiments, the condition is celiac disease or wheat allergy.

[0385] Without being bound by theory, it is believed that the inflammatory response to gluten in the intestine of affected individuals is due to incomplete hydrolysis of gluten, resulting in the formation of toxic (immunotoxic) gluten peptides. The toxicity of gluten (e.g., gliadin and glutenin) is believed to be primarily due to proline- and glutamine-rich peptides produced during incomplete degradation of the protein by human digestive enzymes (including pepsin). Gastric and pancreatic endoproteases are unable to cleave these incompletely degraded toxic or immunogenic peptide byproducts, at least in part because such enzymes lack specificity for proline and / or glutamine. These toxic peptide byproducts include, but are not limited to, "33-mer" and "p31-49" peptides. Toxic peptides are believed to cause a number of intestinal symptoms in susceptible individuals, including intraepithelial lymphocytosis, villous atrophy and / or inflammation. Other proteins present in wheat are also associated with autoimmune responses, including serpins, purinins, alpha-amylase / protease inhibitors, globulins, and farinins.

[0386] Neprosin alone or in combination with other proteases (such as Nepenthes protease I and / or Nepenthes protease II) is able to cleave toxic food peptides into smaller non-toxic peptides, or at least peptides with reduced immunogenicity. Because neprosin and Nepenthes proteases are active over a wide acidic pH range, enzymatic digestion can begin in the acidic environment of the stomach. Administration of neprosin and / or Nepenthes proteases in combination with potentially antigenic food proteins results in a reduced immune response to the antigenic food proteins after ingestion, including a reduction in the infiltration and / or production of intraepithelial lymphocytes in the intestine. Intraepithelial lymphocytes are T cells that are interspersed between epithelial cells in the large and small intestines. Increased T cell counts are an early indicator of inflammation and are potentially associated with gluten intolerance, including celiac disease. When used in combination with food, administration of recombinant neprosin or a composition thereof is able to degrade food protein antigens to a level at which the immune response in the intestine, as measured by IEL infiltration, is attenuated or eliminated. IEL infiltration due to the presence of peptide food antigens is an early biological indicator of sensitivity to food antigens (eg, gluten).

[0387] Thus, in one aspect, the use of recombinant neprosin polypeptides relates to a method for attenuating or preventing an immune response to a food protein antigen in the intestinal tract of a mammal, the method comprising administering an effective amount of a recombinant neprosin polypeptide described herein to a subject in need thereof. In some embodiments, recombinant neprosin is administered alone or in combination with Nepenthes protease I and / or Nepenthes protease II. In some embodiments, the amount of the pharmaceutical composition is effective to attenuate or prevent intestinal IEL infiltration due to the presence of peptide food antigens.

[0388] In some embodiments, the use of a recombinant neprosin polypeptide relates to a method for attenuating or preventing intestinal inflammation caused by the presence of a peptide food antigen in the intestine of a subject, the method comprising administering an effective amount of a recombinant neprosin polypeptide to a subject in need thereof. As described above, intestinal inflammation is characterized by infiltration and / or proliferation of IELs in the intestine. In some embodiments, recombinant neprosin is administered alone or in combination with Nepenthes protease I and / or Nepenthes protease II. In some embodiments, the amount of neprosin, alone or in combination with Nepenthes protease I and / or Nepenthes protease II, is effective to attenuate or prevent intestinal inflammation caused by the presence of a peptide food antigen.

[0389] In some embodiments, the use of a recombinant neprosin polypeptide relates to a method for attenuating or preventing intraepithelial lymphocytosis caused by the presence of a peptide food antigen in the intestine of a subject, the method comprising administering an effective amount of a recombinant neprosin polypeptide to a subject in need thereof. In some embodiments, the recombinant neprosin is administered alone or in combination with Nepenthes protease I and / or Nepenthes protease II. In some embodiments, the amount of neprosin alone or in combination with Nepenthes protease I and / or Nepenthes protease II is effective to inhibit intraepithelial lymphocytosis in the intestine.

[0390] In some embodiments, the use of recombinant neprosin polypeptides relates to a method for attenuating or preventing the generation and / or recruitment of IELs in the intestine due to the presence of peptide food antigens in the intestine of a mammal, the method comprising administering an effective amount of a recombinant neprosin polypeptide to a subject in need thereof. In some embodiments, recombinant neprosin is administered alone or in combination with Nepenthes protease I and / or Nepenthes protease II. In some embodiments, the amount of neprosin alone or in combination with Nepenthes protease I and / or Nepenthes protease II is effective to degrade gluten, thereby attenuating or preventing the generation and / or recruitment of IELs in the intestine.

[0391] In some embodiments, the use of a recombinant neprosin polypeptide relates to a method for reducing a T cell response to a peptide food antigen, the method comprising contacting the peptide food antigen with an effective amount of a recombinant neprosin polypeptide. In some embodiments, the recombinant neprosin is administered alone or in combination with Nepenthes protease I and / or Nepenthes protease II. In some embodiments, the amount of neprosin alone or in combination with Nepenthes protease I and / or Nepenthes protease II is effective to reduce a T cell response to the antigen. In some embodiments, the T cell response in the intestine of a mammal is reduced.

[0392] In some embodiments, the use of a recombinant neprosin polypeptide relates to a method for attenuating or preventing villous atrophy due to the presence of a peptide food antigen in the intestine of a subject, the method comprising administering to a subject in need thereof an effective amount of a recombinant neprosin polypeptide. In some embodiments, the recombinant neprosin is administered alone or in combination with Nepenthes protease I and / or Nepenthes protease II. In some embodiments, the amount of neprosin, alone or in combination with Nepenthes protease I and / or Nepenthes protease II, is effective to degrade gluten to attenuate or prevent villous atrophy in the intestine. In some embodiments, villous atrophy is a result of intestinal inflammation.

[0393] In some embodiments, the use of recombinant neprosin polypeptides relates to a method for treating and / or ameliorating at least one symptom associated with an immune response to the presence of gluten or other antigenic proteins in the intestine of a patient, the method comprising administering to a subject in need thereof an effective amount of a recombinant neprosin polypeptide to treat and / or ameliorate at least one symptom associated with an immune response to the presence of gluten or other antigenic proteins. In some embodiments, such symptoms include, but are not limited to, "foggy mind", depression, anxiety, ADHD-like behavior, abdominal pain, bloating, diarrhea, constipation, headache, migraine, bone or joint pain, chronic fatigue, small intestinal damage, production of tissue transglutaminase (tTG) antibodies, severe acne, vomiting, weight loss, irritability, iron deficiency anemia, arthritis, tingling and numbness in the extremities, infertility, and oral ulcers. In some embodiments, recombinant neprosin is administered alone or in combination with Nepenthes protease I and / or Nepenthes protease II.

[0394] In some embodiments, the use of a recombinant neprosin polypeptide relates to a method for attenuating or preventing manifestations of celiac disease caused by the presence of partially hydrolyzed gluten or wheat protein in the intestine of a patient suffering from celiac disease, the method comprising administering to the patient an effective amount of a recombinant neprosin polypeptide as described herein. In some embodiments, the recombinant neprosin is administered alone or in combination with Nepenthes protease I and / or Nepenthes protease II. In some embodiments, the amount of neprosin, alone or in combination with Nepenthes protease I and / or Nepenthes protease II, is effective to attenuate or prevent manifestations of celiac disease.

[0395] In another aspect, a method for treating gluten intolerance or related conditions such as celiac disease, wheat allergy, gluten sensitivity, and dermatitis herpetiformis in a patient in need thereof is provided, the method comprising treating a food containing gluten or suspected of containing gluten with an effective amount of a recombinant neprosin polypeptide or a composition thereof before it is consumed by the patient. In some embodiments, the food is combined with an effective amount of a recombinant neprosin polypeptide or a composition thereof during its preparation. In some embodiments, the neprosin polypeptide is added after any heating step in the preparation of the food. In some embodiments, the neprosin polypeptide is added before one or more heating steps in the preparation of the food. In some embodiments, the recombinant neprosin is administered alone or in combination with Nepenthes protease I and / or Nepenthes protease II.

[0396] In some embodiments, the use of a recombinant neprosin polypeptide relates to a method for improving the digestibility of protein from food in a subject suffering from an intestinal disorder, the method comprising administering an effective amount of a recombinant neprosin polypeptide to a subject in need thereof. In some embodiments, recombinant neprosin is administered alone or in combination with Nepenthes protease I and / or Nepenthes protease II. In some embodiments, the amount of neprosin, alone or in combination with Nepenthes protease I and / or Nepenthes protease II, is effective to sufficiently degrade protein to improve absorption of protein in the intestine. In some embodiments, the intestinal disorder is Crohn's disease, irritable bowel syndrome, or colitis. In some embodiments, protein absorption from food is increased.

[0397] In some embodiments, recombinant neprosin or a composition thereof is administered to a subject before the subject ingests a potentially antigenic food or protein. In some embodiments, recombinant neprosin or a composition thereof is administered to a subject while the subject ingests a potentially antigenic food or protein. In some embodiments, recombinant neprosin or a composition thereof is administered to a subject after the subject ingests a potentially antigenic food or protein. In some embodiments, recombinant neprosin or a composition thereof is administered to a subject immediately after the subject ingests a potentially antigenic food or protein. In some embodiments, recombinant neprosin or a composition thereof is administered to a subject regardless of the consumption of a potentially antigenic food or protein. In some embodiments, the potentially antigenic protein is gluten. In some embodiments, the potentially antigenic protein is one or more wheat proteins.

[0398] In some embodiments, the subject is a human patient. In some embodiments, the human patient selected for treatment suffers from gluten sensitivity or celiac disease. In some embodiments, the human patient suffers from celiac disease. In some embodiments, the human patient suffers from a disease selected from the group consisting of gluten intolerance, celiac disease, attention deficit hyperactivity disorder, autism, rheumatoid arthritis, fibromyalgia and dermatitis herpetiformis. In some embodiments, the human patient suffers from food allergies. In some embodiments, intestinal antigen protein sensitivity is directly or indirectly related to attention deficit hyperactivity disorder, autism, rheumatoid arthritis, fibromyalgia and / or dermatitis herpetiformis. It is also expected that the use of the compositions of the present invention to remove such antigenic intestinal proteins from the intestine will have a positive effect on attention deficit hyperactivity disorder, autism, rheumatoid arthritis, fibromyalgia and / or dermatitis herpetiformis.

[0399] In some embodiments, the pharmaceutical composition is administered orally before, during, or immediately after consumption of a gluten-containing or other immunogenic food.

[0400] In some embodiments, the recombinant neprosin polypeptide or a composition thereof is administered to a subject before the subject ingests food containing gluten or suspected of containing gluten. In some embodiments, the neprosin polypeptide or a composition thereof is administered during a time period during which the enzyme is at least partially effective (e.g., at least about 10%, 20%, 50%, 70%, 90% of the initial activity) to degrade gluten in food that the subject will ingest. In some embodiments, the neprosin polypeptide or a composition thereof is administered no more than about 4 hours, 3 hours, 2 hours, 1 hour, or 30 minutes before the subject ingests food.

[0401] In some embodiments, the neprosin polypeptide or composition thereof is administered to the subject while the subject is ingesting a potentially immunogenic food. In some embodiments, the neprosin polypeptide or composition thereof is administered with food. In some embodiments, the neprosin polypeptide or composition thereof is administered separately from food.

[0402] In some embodiments, the neprosin polypeptide or its composition is administered to the subject shortly after the subject ingests a potentially immunogenic food. In some embodiments, the neprosin polypeptide or its composition is administered during a time period when at least a portion (e.g., at least about 10%, 20%, 50%, 70%, 90%) of the antigen in the food is still in the stomach of the subject. In some embodiments, the neprosin polypeptide or its composition is administered no more than 4 hours, 3 hours, 2 hours, 1 hour, or 30 minutes after the subject ingests the food.

[0403] Typically, the neprosin polypeptide or composition thereof is administered in an amount that is safe and sufficient to produce the desired detoxification effect of the peptide food antigen. The dosage of the neprosin polypeptide or composition thereof may vary depending on factors such as the specific enzyme or combination administered, the subject's sensitivity to food, the amount and type of antigen-containing food ingested, the pharmacodynamic properties of the enzyme, the mode of administration, the age, health status and weight of the recipient, the nature and extent of the symptoms, the frequency of treatment and the type of concurrent treatment (if any), and the clearance rate of the enzyme. A person skilled in the art can determine an appropriate dosage based on the above factors. The neprosin polypeptide or composition thereof may be initially administered at an appropriate dosage that can be adjusted as needed based on the clinical response. In vitro assays may optionally be used to help identify the optimal dosage range. The precise dosage to be employed in the formulation will also depend on the route of administration and / or the severity of the disease or disorder, and should be determined according to the judgment of the practitioner and the circumstances of each subject.

[0404] In some embodiments, the dosage or dosing regimen for adult subjects can be adjusted proportionally for children and infants, and also for other administrations or other forms, such as proportional to molecular weight or immune response. Administration or treatment can be repeated at appropriate intervals at the discretion of the physician.

[0405] In some embodiments, the effective amount of a recombinant neprosin polypeptide or composition thereof is between about 1 mg and about 1 g. In some embodiments, the effective amount is between about 1 mg and about 10 mg. In some embodiments, the effective amount is less than about 5 mg / day. In some embodiments, the effective amount is between about 10 mg and about 1 g. In some embodiments, the effective amount is between about 20 mg and about 1 g. In one embodiment, the effective amount is between about 30 mg and about 1 g. In some embodiments, the effective amount is between about 40 mg and about 1 g. In some embodiments, the effective amount is between about 50 mg and about 1 g. In some embodiments, the effective amount is between about 60 mg and about 1 g. In some embodiments, the effective amount is between about 70 mg and about 1 g. In some embodiments, the effective amount is between about 80 mg and about 1 g. In some embodiments, the effective amount is between about 100 mg and about 1 g. In some embodiments, the effective amount is between about 500 mg and about 1 g. In some embodiments, the effective amount is between about 1 mg and about 500 mg. In some embodiments, the effective amount is between about 1 mg and about 250 mg. In some embodiments, the effective amount is between about 1 mg and about 200 mg. In some embodiments, the effective amount is between about 1 mg and about 100 mg. In some embodiments, the effective amount is between about 1 mg and about 90 mg. In some embodiments, the effective amount is between about 1 mg and about 80 mg. In some embodiments, the effective amount is between about 1 mg and about 70 mg. In some embodiments, the effective amount is between about 1 mg and about 60 mg. In some embodiments, the effective amount is between about 1 mg and about 50 mg. In some embodiments, the effective amount is between about 1 mg and about 40 mg. In some embodiments, the effective amount is between about 1 mg and about 30 mg. In some embodiments, the effective amount is between about 1 mg and about 20 mg. In some embodiments, the effective amount is between about 1 mg and about 5 mg. In some embodiments, the effective amount is between about 1 mg and about 4 mg. In some embodiments, the effective amount is between about 1 mg and about 3 mg. This includes any value within any of these ranges, inclusive, and sub-ranges between any two of these values.

[0406] In some embodiments, the recombinant neprosin polypeptide is administered at a dose of about 0.001 mg to about 1000 mg of enzyme per kg body weight per day, or about 1 mg to about 100 g per dose for an average person. In some embodiments, the recombinant neprosin polypeptide can be administered at 0.001 mg, 0.01 mg, 0.1 mg, 1 mg, 5 mg, 10 mg, 50 mg, 100 mg, 500 mg, or 1000 mg per kg body weight per day, and a range (including endpoints) between any two of these values. In some embodiments, the recombinant neprosin polypeptide can be administered at 1 mg, 10 mg, 100 mg, 200 mg, 500 mg, 700 mg, 1 g, 10 g, 20 g, 50 g, 70 g, 100 g, and a range (including endpoints) between any two of these values ​​per dose. In some embodiments, the recombinant neprosin polypeptide can be administered once, twice, three times, etc., per day, depending on the number of times the subject ingests food containing the antigenic protein and / or how much such food is consumed. The amounts of recombinant neprosin polypeptide recited herein may relate to the total enzyme or to each individual enzyme in the composition.

[0407] In some embodiments, the amount of recombinant neprosin polypeptide administered is based on the amount (or approximate amount) of the substrate (e.g., gluten and / or other proteins or potentially antigenic proteins) consumed / to be consumed. In some embodiments, about 1 mg to about 1 g of enzyme is administered per 1 g of substrate. In some embodiments, about 5 mg to about 1 g of enzyme is administered per 1 g of substrate. In some embodiments, about 10 mg to about 1 g of enzyme is administered per 1 g of substrate. In some embodiments, about 100 mg to about 1 g of enzyme is administered per 1 g of substrate. In some embodiments, about 1 mg to about 500 mg of enzyme is administered per 1 g of substrate. In some embodiments, about 1 mg to about 250 mg of enzyme is administered per 1 g of substrate. In some embodiments, about 1 mg to about 100 mg of enzyme is administered per 1 g of substrate. In some embodiments, about 1 mg to about 100 mg of enzyme is administered per 1 g of substrate. In some embodiments, about 1 mg to about 10 mg of enzyme is administered per 1 g of substrate. This includes any value within these ranges (including the endpoints), as well as subranges between any two of these values.

[0408] In a preferred embodiment, the ratio of substrate to enzyme is between about 10: 1 and about 1000: 1. In some embodiments, the ratio of substrate to enzyme is between about 10: 1 and about 100: 1.

[0409] In some embodiments, the ratio of substrate (total protein) to enzyme (neprosin or a combination of pitcher plant enzymes) administered is between about 1:1 and about 15000:1. In some embodiments, the ratio of substrate to enzyme is between about 10:1 and about 15000:1. In some embodiments, the ratio of substrate to enzyme is between about 100:1 and about 15000:1. In some embodiments, the ratio of substrate to enzyme is between about 500:1 and about 15000:1. In some embodiments, the ratio of substrate to enzyme is between about 1000:1 and about 15000:1. In some embodiments, the ratio of substrate to enzyme is between about 5000:1 and about 15000:1. In some embodiments, the ratio of substrate to enzyme is between about 100000:1 and about 15000:1. In some embodiments, the ratio of substrate to enzyme is between about 1:1 and about 10000:1. In some embodiments, the ratio of substrate to enzyme is between about 10: 1 and about 10000: 1. In some embodiments, the ratio of substrate to enzyme is between about 100: 1 and about 10000: 1. In some embodiments, the ratio of substrate to enzyme is between about 500: 1 and about 10000: 1. In some embodiments, the ratio of substrate to enzyme is between about 1000: 1 and about 10000: 1. In some embodiments, the ratio of substrate to enzyme is between about 5000: 1 and about 10000: 1. In some embodiments, the ratio of substrate to enzyme is between about 1: 1 and about 5000: 1. In some embodiments, the ratio of substrate to enzyme is between about 10: 1 and about 5000: 1. In some embodiments, the ratio of substrate to enzyme is between about 100: 1 and about 5000: 1. In some embodiments, the ratio of substrate to enzyme is between about 500: 1 and about 5000: 1. In some embodiments, the ratio of substrate to enzyme is between about 1000: 1 and about 5000: 1. In some embodiments, the ratio of substrate to enzyme is between about 1: 1 and about 1000: 1. In some embodiments, the ratio of substrate to enzyme is between about 10: 1 and about 1000: 1. In some embodiments, the ratio of substrate to enzyme is between about 100: 1 and about 1000: 1. In some embodiments, the ratio of substrate to enzyme is between about 1: 1 and about 500: 1. In some embodiments, the ratio of substrate to enzyme is between about 10: 1 and about 500: 1. In some embodiments, the ratio of substrate to enzyme is between about 100: 1 and about 500: 1. In some embodiments, the ratio of substrate to enzyme is between about 1: 1 and about 100: 1. In some embodiments, the ratio of substrate to enzyme is between about 10: 1 and about 100: 1. In some embodiments, the ratio of substrate to enzyme is between about 10: 1 and about 100: 1. This includes any value (including endpoints) within any of these ranges, as well as subranges between any two of these values.

[0410] In some embodiments, total protein can be, for example, the total protein consumed in a given meal or the total protein consumed in a specific time period (e.g., within 1 hour, 2 hours, or 3-24 hours). In some embodiments, total protein is the sum of all proteins consumed by a subject within a 1 hour time period. In some embodiments, total protein is the sum of all proteins consumed by a subject within a 2 hour time period. In some embodiments, total protein is the sum of all proteins consumed by a subject within a 3 hour time period. In some embodiments, total protein is the sum of all proteins consumed by a subject within a 4 hour time period. In some embodiments, total protein is the sum of all proteins consumed by a subject within a 5 hour time period. In some embodiments, total protein is the sum of all proteins consumed by a subject within a 10 hour time period. In some embodiments, total protein is the sum of all proteins consumed by a subject within a 12 hour time period. In some embodiments, total protein is the sum of all proteins consumed by a subject within a 15 hour time period. In some embodiments, total protein is the sum of all proteins consumed by a subject within a 20 hour time period. In some embodiments, total protein is the sum of all proteins consumed by a subject within a 24 hour time period.

[0411] In some embodiments, regardless of whether the patient ingests (e.g., intentionally ingests) food containing potentially immunogenic proteins, the pharmaceutical composition is administered. In some embodiments, the pharmaceutical composition is administered as needed, for example, before, during, and / or after a meal that may be contaminated with potentially immunogenic proteins or in which the content of potentially immunogenic proteins is unknown. In some embodiments, the pharmaceutical composition is administered regularly. In some embodiments, the pharmaceutical composition is administered at least once a day. In some embodiments, the pharmaceutical composition is administered twice, three times, four times, or more a day. In some embodiments, the pharmaceutical composition is administered in conjunction with each meal and / or snack (e.g., before, during, or after each meal and / or snack). In some embodiments, the pharmaceutical composition is included as a part of a sustained release formulation, in which there is a continuous release of an enzyme to allow intermittent snacking, etc., without considering the antigenic protein content of the food.

[0412] In some embodiments and as discussed herein, the recombinant neprosin polypeptide can be administered as the sole active agent, or it can be administered in combination with other agents (simultaneously, sequentially or separately, or by co-formulation), including other compounds that exhibit the same or similar therapeutic activity and are determined to be safe and effective for such combined administration. In some embodiments, as described above, the pharmaceutical composition is administered with an additional enzyme such as pepsin, an aspartic protease (such as pepsin, pepsinogen, or those described by Chen et al., Gene, 2009, 442: 108-118) and an enzyme such as another prolyl endopeptidase (PEP), dipeptidyl peptidase IV (DPP IV) and dipeptidyl carboxypeptidase (DCP) or cysteine ​​protease B (see, e.g., U.S. Patent No. 7,910,541), as described herein. In some embodiments, the additional enzyme is administered in the form of a bacterium that produces and / or secretes the additional enzyme. In some embodiments, the bacteria are engineered to produce and / or secrete neprosin alone or in combination with Nepenthes protease I and / or Nepenthes protease II.

[0413] In some embodiments, the pharmaceutical composition is administered to a subject together with another therapeutic agent that is not a protease. Non-limiting examples of agents that can be administered with the pharmaceutical composition include inhibitors of tissue transglutaminase, anti-inflammatory agents such as amylases, glucoamylases, endopeptidases, HMG-CoA reductase inhibitors (e.g., compactin, lovastatin, simvastatin, pravastatin, and atorvastatin), leukotriene receptor antagonists (e.g., montelukast and zafirlukast), COX-2 inhibitors (e.g., celecoxib and rofecoxib), p38 MAP kinase inhibitors (e.g., BIRB-796); mast cell stabilizers such as sodium cromoglycate (cromolyn), pimilast, proxicromil, repirinast, doxantrazole, amlexanox, nedocromil and probicromil, anti-ulcer agents, anti-allergic agents such as antihistamines (e.g., acrivastine, cetirizine, desloratadine, ebastine, fexofenadine, levocetirizine, loratadine and mizolastine), inhibitors of transglutaminase 2 (TG2), anti-TNF α agents and antibiotics. In some embodiments, the additional agent is a probiotic. Probiotics include, but are not limited to, species and strains of the genus Lactobacillus, Saccharomyces, Bacillus or Bifidobacterium. In some embodiments, the other agent is elafin. In some embodiments, the additional agent is administered in the form of a bacterium that produces and / or secretes the additional agent. Example

[0414] The following examples, including the experiments and results obtained, are provided for illustrative purposes only and should not be construed as limiting the present invention. Example 1 Expression of neprosin in mammalian cells

[0415] On the day of transfection, determine the viable cell density and percent viability of the prepared cells. Dilute the cells to 3 × 10 cells / mL using pre-warmed Expi293™ Expression Medium. 6The final viable cell density was ≥ 95% viable cells / mL and the percent viability should be ≥ 95%. Gently swirl the culture flask to mix the cells in preparation for transfection. Transient transfection was performed using the recommended protocol from the ExpiFectamine™ 293 Transfection Kit. Check the viable cell density and percent viability every two days. Harvest the culture when the viable cell density is less than half of the peak maximum, or when the percent viability of the viable cells is less than 70%. Neprosin I (Npr1) is secreted into the culture medium and the supernatant is harvested on day 5 after transfection. The entire culture was centrifuged at 4,000 RCF for 30 min in a benchtop refrigerated centrifuge in a 50 mL falcon tube and the supernatant was filtered using a 0.22-μm filter. Selected samples were purified. Example 2 Activation of recombinant neprosin and its activity towards gliadin substrates

[0416] Formation of active neprosin protease peptides An 80 µL aliquot of ~40 µM Expi293 or ExpiCHO purified Npr1 (prepared from cells expressing mSP-His-Npr1; SEQ ID NO: 40) was added to a 3.5K MWCOSlide-a-Lyzer MINI dialysis unit (Thermo Scientific, USA) containing 14 mL of 100 mM glycine (pH 2.5). The dialysis unit was incubated at 37°C, 250 rpm. At the incubation time point, 4 μL of enzyme was removed and diluted 1:5 into 1× McIlvaine buffer (pH 7.5). Immediately after dilution, the sample was mixed 1:1 with 2× SDS-PAGE sample buffer. The sample in SDS-PAGE buffer was heated at 95°C for 10 min, and 10 μL was loaded onto a 12-well NuPAGE gel (4%-12% Bis-Tris). SDS-PAGE was run at 200 V for 35 min, and the gel was stained with Coomassie blue. Figure 2 Shown in.

[0417] pH activation curve of neprosin protease peptide A 50 μL aliquot of ~8 µM purified Npr1 (mSP-His-Npr1; SEQ ID NO: 40) was added to a 3.5 K MWCO Slide-a-Lyzer mini-dialysis device containing 14 mL of 1× MacIlvaine buffer or 1× simulated gastric fluid (SGF; 7 mM KCl, 1 mM KH2PO4, 47 mM NaCl, 0.1 mM MgCl2(H2O)6, 0.075 mM CaCl2(H2O)2) ± 0.8 g / L pepsin at the indicated pH (i.e., pH 2.5, 3, 3.5, 4, 4.5, or 5). The dialysis device was incubated at 37°C, 250 rpm. At 60 min, 50 μL of enzyme sample was recovered and placed in a PCR tube. 10 μL of these samples were immediately mixed 1:1 with 2× SDS-PAGE sample buffer. The remaining sample was placed at room temperature for 5 h without shaking and then mixed with sample buffer as described above. The sample in SDS-PAGE buffer was heated at 95°C for 10 min and 10 μL was loaded onto a 12-well NuPAGE gel (4%-12% Bis-Tris). SDS-PAGE was run at 200 V for 35 min and the gel was stained with Coomassie blue. The results are shown in Figure 3 Shown in.

[0418] Activity of activated neprosin protease towards gliadin substrates A ~1.2 mg / mL solution of the dissolved gliadin stock (previously prepared and frozen) was centrifuged at 15,000 rpm and the soluble fraction was transferred to a fresh tube. The soluble fraction was diluted 1:10 in ddH2O to give a ~0.12 mg / mL working stock. In a PCR tube, a 50 μL aliquot of ~0.12 mg / mL gliadin was added to 40 μL of 100 mM sodium acetate buffer (pH 4). The neprosin enzyme aliquot (10 µL of 0.06 mg / mL or 0.006 mg / mL enzyme preparation) was added to start the reaction and incubated at 37°C, 900 rpm for 5 min or 30 min. The reaction was quenched by placing the reaction tube in a preheated (95°C) thermal cycler and heating at 95°C for 10 min. The samples were diluted 15,000-fold before analysis using the RIDASCREEN® Gliadin Competitive ELISA. The sample without enzyme was used to calculate the percentage of remaining gliadin. Figure 4 As shown in , the neprosin preparation activated by treatment at acidic pH exhibited significantly higher proteolytic activity compared to the inactivated neprosin polypeptide prepared from cells expressing mSP-His-Npr1. Example 3 Transfection and expression of neprosin in Pichia pastoris cells

[0419] Transfection of Pichia pastoris cells with neprosin gene 200 ng of Pme1 linearized DNA was added to 30 μL of Pichia BG10 electrocompetent cells and mixed gently. The cell and DNA mixture was transferred to a cooled 1 mm electroporation cuvette and electroporated using 25 μF, 200 Ω, and 1150 V (EXM 630, BTX). 1 mL of Pichia electroporation recovery solution (YPD: 1M sorbitol, 1:1 (v / v)) was added to the cuvette, gently mixed with the cell solution, and transferred to different test tubes. The electroporated cells were incubated at 20°C at 100 rpm for 3 h. Then 100 μL of the incubated cells were spread onto YPD+800 μg / mL G418 agar plates in multiple dilutions to obtain single colonies and incubated at 30°C for 48 h. To generate glycerol stocks, single colonies were picked and used to inoculate 2 ml of BMY medium (BMGY) containing 1.5% glycerol and grown at 30°C, 250 rpm for 48 h. Cells were stored in 25% glycerol at -80°C.

[0420] Expression of neprosin in Pichia pastoris Use the glycerol stock of Pichia cells containing the neprosin (Npr1) gene to streak on YPD+800 μg / mL G418 agar plates. The plates were incubated at 30°C for 48 h. A single colony from the agar plate was used to inoculate 50 mL BMGY. The starter culture was incubated at 30°C for 24 h at 200 rpm and then used to inoculate (1:50) a shake flask containing BMGY. The culture was allowed to grow for 48 h at 30°C, 250 rpm. Cells were collected by centrifugation and then resuspended in BMY medium (BMMY) containing 2% methanol to induce protein expression. Secreted Npr1 was obtained by harvesting the supernatant (4,000 rpm, 20 min) after 24 h. The supernatant was filtered using a 0.45-μm filter and purified. SDS-PAGE analysis of WT, N216A and N216A, N324ANpr1 (SEQ ID NOs: 56, 60 and 66, respectively) Figure 5 Shown in. Example 4 Activation of recombinant neprosin from Pichia pastoris and its activity towards gliadin substrates

[0421] Formation of active neprosin protease peptides WT Npr1, N216A Npr1, or N216A / N324A Npr1 (SEQ ID NOs: 54, 60, and 66, respectively) prepared from Pichia pastoris cells were added to a 10K MWCO Slide-A-Lyzer dialysis cassette (Thermo Scientific, USA) and dialyzed against 100 mM glycine (pH 2.5) for 4 h at 37°C with gentle stirring. The dialyzed protein sample was mixed 1:1 with 2× SDS-PAGE sample buffer and heated at 95°C for 10 min. 10 μL of the sample was loaded onto a 10-well NuPAGE gel (4%-12% Bis-Tris). SDS-PAGE was run at 200 V for 35 min, and the gel was stained with Coomassie blue. The results are shown in Figure 6 Activated WT neprosin appears as two glycosylated polypeptides of approximately 29 kDa and 35 kDa. Activated N216A neprosin and N216A / N324A neprosin appear as a monoglycosylated species of approximately 29 kDa.

[0422] The activated WT neprosin band was subjected to N-terminal sequencing using in-gel digestion LC-MS / MS analysis (Thermo Orbitrap Exploris 240), identifying P199-S200 (SEQ ID NO: 54) as the cleavage site between the propeptide and activated Npr1. This analysis also confirmed the identity of the two protein bands as activated Npr1, and their molecular weight differences were due to differences in glycosylation of mannose chain lengths.

[0423] Activity of activated Pichia pastoris neprosin towards gliadin substrates A ~32 mg / mL solution of the dissolved gliadin stock (previously prepared and frozen) was centrifuged at 15,000 rpm and the soluble fraction was transferred to a fresh tube. The soluble fraction was diluted 1:1 in ddH2O to give a ~16 mg / mL working stock. 10 µL of ~16 mg / mL gliadin was added to 80 µL of 10× simulated gastric fluid solution (SGF: McIlvaine buffer, pH 3, 8 mg / mL pepsin) in a PCR tube. The reaction was started by adding 10 μL of 0.016 mg / mL activated Pichia WT Npr1 enzyme preparation and incubated at 37°C, 900 rpm for 60 min, collecting samples at 0 min, 5 min, 15 min, 30 min, and 60 min. The reaction was quenched by placing the reaction tube in a preheated (95°C) thermal cycler and heating at 95°C for 10 min. Samples were diluted 1,000-fold before analysis using the RIDASCREEN® Gliadin Competitive ELISA (R-Biopharm AG, Germany). Samples prepared in CHO cells without neprosin enzyme or activated neprosin protein were run in parallel for comparison...

Claims

1. A recombinant neprosin polypeptide comprising an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence of a prepropolypeptide, propolypeptide, mature polypeptide or polypeptide corresponding to SEQ ID NO: 1, wherein the amino acid sequence comprises a substitution with an amino acid other than asparagine (N) at at least amino acid position 68, 145, 152 or 253, or a combination thereof, and the equivalent thereof, wherein the amino acid positions are relative to the reference sequence corresponding to SEQ ID NO:

1.

2. The recombinant neprosin polypeptide according to claim 1, wherein the amino acid sequence of the recombinant neprosin polypeptide comprises a substitution at least at amino acid position 68 with an amino acid other than asparagine (N).

3. The recombinant neprosin polypeptide according to claim 1, wherein the amino acid sequence of the recombinant neprosin polypeptide comprises a substitution with an amino acid other than asparagine (N) at least at amino acid position 152.

4. The recombinant neprosin polypeptide according to claim 1, wherein the amino acid sequence of the recombinant neprosin polypeptide comprises a substitution with an amino acid other than asparagine (N) at least at amino acid position 253.

5. The recombinant neprosin polypeptide according to claim 1, wherein the amino acid sequence of the recombinant neprosin polypeptide comprises a substitution at least at amino acid position 145 with an amino acid other than asparagine (N).

6. The recombinant neprosin polypeptide according to any one of claims 1 to 5, wherein the signal sequence of the prepropolypeptide is replaced by a signal sequence functional in mammalian cells, insect cells or fungal cells, or wherein the propolypeptide further comprises a signal sequence functional in mammalian cells, insect cells or fungal cells.

7. A recombinant neprosin polypeptide comprising an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence corresponding to residues 105 to 356 of SEQ ID NO: 8, or to a reference sequence corresponding to SEQ ID NO: 8, wherein the amino acid sequence comprises a substitution with an amino acid other than asparagine (N) at at least amino acid position 44, 121, 128 or 229, or a combination thereof, wherein the amino acid positions are relative to SEQ ID NO:

8.

8. The recombinant neprosin polypeptide according to claim 7, wherein the amino acid sequence of the recombinant neprosin polypeptide comprises a substitution at least at amino acid position 44 with an amino acid other than asparagine (N).

9. The recombinant neprosin polypeptide according to claim 8, wherein the amino acid sequence of the recombinant neprosin polypeptide comprises SEQ ID NO:

10.

10. The recombinant neprosin polypeptide of claim 7, wherein the amino acid sequence of the recombinant neprosin polypeptide comprises a substitution with an amino acid other than asparagine (N) at least at amino acid position 121.

11. The recombinant neprosin polypeptide of claim 10, wherein the amino acid sequence of the recombinant neprosin polypeptide comprises residues 105 to 356 of SEQ ID NO: 12, or comprises SEQ ID NO:

12.

12. The recombinant neprosin polypeptide of claim 7, wherein the amino acid sequence of the recombinant neprosin polypeptide comprises a substitution at least at amino acid position 128 with an amino acid other than asparagine (N).

13. The recombinant neprosin polypeptide of claim 12, wherein the amino acid sequence of the recombinant neprosin polypeptide comprises residues 105 to 356 of SEQ ID NO: 14, or comprises SEQ ID NO:

14.

14. The recombinant neprosin polypeptide of claim 7, wherein the amino acid sequence of the recombinant neprosin polypeptide comprises a substitution at least at amino acid position 229 with an amino acid other than asparagine (N).

15. The recombinant neprosin polypeptide of claim 14, wherein the amino acid sequence of the recombinant neprosin polypeptide comprises residues 105 to 356 of SEQ ID NO: 16, or comprises SEQ ID NO:

16.

16. The recombinant neprosin polypeptide of claim 7, wherein the amino acid sequence of the recombinant neprosin polypeptide comprises substitutions with amino acids other than asparagine (N) at least at amino acid positions 121 and 229.

17. The recombinant neprosin polypeptide of claim 16, wherein the amino acid sequence of the recombinant neprosin polypeptide comprises residues 105 to 356 of SEQ ID NO: 18, or comprises SEQ ID NO:

18.

18. The recombinant neprosin polypeptide of claim 7, wherein the amino acid sequence of the recombinant neprosin polypeptide comprises substitutions with amino acids other than asparagine (N) at least at amino acid positions 44, 128, and 229.

19. The recombinant neprosin polypeptide of claim 18, wherein the amino acid sequence of the recombinant neprosin polypeptide comprises residues 105 to 356 of SEQ ID NO: 20, or comprises SEQ ID NO:

20.

20. The recombinant neprosin polypeptide of claim 7, wherein the amino acid sequence of the recombinant neprosin polypeptide comprises substitutions with amino acids other than asparagine (N) at least at amino acid positions 44, 121, 128, and 229.

21. The recombinant neprosin polypeptide of claim 20, wherein the amino acid sequence of the recombinant neprosin polypeptide comprises residues 105 to 356 of SEQ ID NO: 22, or comprises SEQ ID NO:

22.

22. The recombinant neprosin polypeptide of claim 7, wherein the amino acid sequence of the recombinant neprosin polypeptide comprises substitutions with amino acids other than asparagine (N) at least at amino acid positions 44 and 229.

23. The recombinant neprosin polypeptide of claim 20, wherein the amino acid sequence of the recombinant neprosin polypeptide comprises residues 105 to 356 of SEQ ID NO: 32, or comprises SEQ ID NO:

32.

24. The recombinant neprosin polypeptide according to any one of claims 7 to 23, wherein the amino acid sequence of the recombinant neprosin polypeptide further comprises a signal sequence.

25. The recombinant neprosin polypeptide of claim 24, wherein the signal sequence is functional in mammalian cells, insect cells or fungal cells.

26. The recombinant neprosin polypeptide of claim 25, wherein the signal sequence is a mammalian signal sequence.

27. The recombinant neprosin polypeptide of claim 25, wherein the signal sequence is a fungal signal sequence.

28. A recombinant neprosin polypeptide comprising an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence corresponding to residues 105 to 356 of SEQ ID NO: 8, or to a reference sequence corresponding to SEQ ID NO: 8, wherein the amino acid sequence further comprises a signal sequence that is functional in a mammalian cell, an insect cell, or a fungal cell.

29. The recombinant polynucleotide of claim 28, wherein the amino acid sequence of the recombinant neprosin polypeptide comprises residues 105-356 of SEQ ID NO: 8, or comprises SEQ ID NO:

8.

30. A recombinant polynucleotide comprising a polynucleotide sequence encoding the recombinant neprosin polypeptide of any one of claims 1-29.

31. The recombinant polynucleotide of claim 30, wherein the polynucleotide sequence is codon-optimized for expression in mammalian cells, insect cells, or fungal cells.

32. The recombinant polynucleotide of claim 30, comprising a polynucleotide sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more sequence identity to a reference polynucleotide sequence corresponding to nucleotide residues 313 to 1068 of SEQ ID NO: 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, or 33, or a sequence corresponding to SEQ ID NO: 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, or 33, wherein the recombinant polynucleotide encodes a neprosin polypeptide.

33. The recombinant polynucleotide of claim 30, comprising nucleotide residues 313 to 1068 comprising SEQ ID NO: 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31 or 33, or a polynucleotide sequence comprising SEQ ID NO: 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31 or 33.

34. An expression vector comprising the recombinant polynucleotide according to any one of claims 30-33.

35. The expression vector of claim 34, wherein the recombinant polynucleotide is operably linked to a promoter.

36. The expression vector of claim 35, wherein the promoter is functional in mammalian cells, insect cells or fungal cells.

37. A host cell comprising the expression vector of any one of claims 34-36, wherein the host cell is a eukaryotic cell.

38. The host cell of claim 37, wherein the eukaryotic cell is a mammalian cell, an insect cell or a fungal cell.

39. The host cell of claim 38, wherein the host cell is a mammalian cell, wherein the mammalian cell is a human cell or a rodent cell.

40. The host cell of claim 38, wherein the host cell is a fungal cell.

41. The host cell of claim 40, wherein the fungal cell is a Saccharomyces or Pichia.

42. The host cell of claim 41, wherein the fungal cell is Saccharomyces cerevisiae or Pichia pastoris.

43. A method for expressing recombinant neprosin, comprising culturing the host cell of any one of claims 37-42 under suitable culture conditions such that the recombinant neprosin polypeptide is expressed.

44. The method of claim 43, wherein the host cell is a mammalian cell.

45. The method of claim 44, wherein the mammalian cell is a rodent cell or a human cell.

46. ​​The method of any one of claims 44-45, wherein the mammalian cell is an Expi293 cell, 293F, 293E, 293T, HeLa cell, U2OS, A549, HT1080, CAD, P19, NIH 3T3, L929, Hek 293 cell, COS, Vero, NSO cell, Sp2 / 0 cell, DUKX-X11, MCF-7, Y79, SO-Rb50, Hep G2, J558L or CHO cell.

47. The method of any one of claims 44-46, wherein the polynucleotide sequence encoding the recombinant neprosin polypeptide is codon-optimized for expression in mammalian cells.

48. The method of claim 43, wherein the host cell is an insect cell.

49. The method of claim 48, wherein the insect cell is a lepidopteran or dipteran insect cell.

50. The method of claim 48, wherein the insect cell is a Sf9 cell, a Sf21 cell, a BTI-TN-5B1-4 (High Five) cell, or a Schneider 2 cell.

51. The method of any one of claims 48-50, wherein the polynucleotide sequence encoding the recombinant neprosin is codon-optimized for expression in insect cells.

52. The method of claim 43, wherein the host cell is a fungal cell.

53. The method of claim 52, wherein the fungal cell is a filamentous fungus or a yeast cell.

54. The method of claim 52, wherein the fungal cell is Saccharomyces cerevisiae, Pichia pastoris, Yarrowia lipolytica, Kluyveromyces marxianus, Kluyveromyces lactis, Aspergillus niger, Aspergillus oryzae, Aspergillus fumigatus, Trichodermareesei, Neurospora crassa, Mucor circinelloides, Penicillium chrysogenum, T. reesei, Trichoderma harzianum, or Myceliophthora thermophile.

55. The method of any one of claims 52-54, wherein the polynucleotide sequence encoding the recombinant neprosin is codon-optimized for expression in the fungal cell.

56. The method of any one of claims 43-55, wherein the recombinant neprosin polypeptide expressed is a preproenzyme / prepropolypeptide of the neprosin polypeptide.

57. The method of any one of claims 43-55, wherein the recombinant neprosin polypeptide expressed is a mature neprosin polypeptide.

58. The method of any one of claims 43-55, wherein the recombinant neprosin polypeptide expressed is a zymogen / propolypeptide of the neprosin polypeptide.

59. The method of any one of claims 57-58, wherein the expressed recombinant neprosin polypeptide is secreted.

60. The method of any one of claims 43-59, wherein the expressed recombinant neprosin polypeptide is glycosylated.

61. The method of any one of claims 43-59, wherein the expressed recombinant neprosin polypeptide is non-glycosylated.

62. The method of any one of claims 43-61, wherein the expressed recombinant neprosin polypeptide is expressed in a soluble form.

63. The method of any one of claims 43-62, wherein the expressed neprosin polypeptide comprises residues 105 to 356 comprising SEQ ID NO: 8, 10, 12, 14, 16, 18, 18, 20, 22, 24, 26, 28, 30, 32, or 34, or an amino acid sequence comprising SEQ ID NO: 8, 10, 12, 14, 16, 18, 18, 20, 22, 24, 26, 28, 30, 32, or 34.

64. The method of any one of claims 43-63, further comprising the step of isolating the expressed recombinant neprosin polypeptide.

65. The method of any one of claims 43-64, further comprising the step of purifying the expressed recombinant neprosin polypeptide.

66. A recombinant neprosin polypeptide produced by the method of any one of claims 43-65.

67. A method for preparing a mature neprosin polypeptide, comprising treating a neprosin prepropolypeptide or propolypeptide of a recombinant neprosin polypeptide of any one of claims 1-33 or a neprosin prepropolypeptide or propolypeptide prepared by the method of any one of claims 43-65 in vitro to a pH of about 2 to about 6 under suitable conditions for forming the mature neprosin polypeptide.

68. The method of claim 67, wherein the pH is from about 2 to about 4.

5.

69. The method of claim 67 or 68, wherein the pH is about 2 to about 3.

70. The method of any one of claims 67-69, wherein the mature neprosin polypeptide has a molecular weight of about 25.5 kDa to about 29 kDa according to SDS-PAGE.

71. A pharmaceutical composition comprising a propolypeptide or a mature neprosin polypeptide of the recombinant neprosin polypeptide of any one of claims 1-33, or a propolypeptide or a mature neprosin polypeptide prepared by the method of any one of claims 43-65, or a mature neprosin polypeptide prepared by the method of any one of claims 67-70.

72. The pharmaceutical composition of claim 71, further comprising a pharmaceutically acceptable carrier, excipient or diluent.

73. A method for treating a disease or condition associated with gluten intolerance, comprising administering to a subject in need thereof an effective amount of a propolypeptide or a proteolytically active mature neprosin polypeptide of any one of claims 1-33 or a pharmaceutical composition of claim 71 or 72.

74. The method of claim 73, wherein the neprosin polypeptide or pharmaceutical composition thereof is administered before, simultaneously with, or after consumption of a gluten-containing food.

75. The method of claim 74, wherein the neprosin polypeptide or pharmaceutical composition thereof is administered prior to consumption of the gluten-containing food.

76. The method of claim 74, wherein the neprosin polypeptide or pharmaceutical composition thereof is administered concurrently with consumption of the gluten-containing food.

77. The method of claim 76, wherein the neprosin polypeptide or pharmaceutical composition thereof is added to the gluten-containing food prior to consumption.

78. The method of claim 74, wherein the neprosin polypeptide or pharmaceutical composition thereof is administered after consumption of the gluten-containing food.

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