Recombinant Neprosin Polypeptide and Method of Expression

Abstract

The present disclosure relates to methods for expressing recombinant nepocin polypeptides, host cells and expression vectors for expressing recombinant nepocin polypeptides, and methods of using the expressed nepocin polypeptides for therapeutic and other purposes. The present disclosure provides, in particular, methods for expressing recombinant nepocin polypeptides that result in efficient expression of soluble forms of nepocin, and the use of recombinant nepocin polypeptides in the treatment of diseases or conditions associated with gluten intolerance such as celiac disease.

Description

Technical Field

[0001] Cross - Reference to Related Applications This application claims the benefit under 35 U.S.C. § 119(e) of U.S. Provisional Patent Application No. 63 / 340,411, filed May 10, 2022, and U.S. Provisional Patent Application No. 63 / 356,181, filed Jun. 28, 2022, the entire contents of each of which are incorporated herein by reference.

[0002] Reference to a Sequence Listing, Table, or Computer Program A sequence listing, having a file size of 187,597 bytes and named CX7 - 229WO3_ST26.txt, created on May 10, 2023, and submitted herewith in connection with this specification, is part of this specification and is incorporated herein by reference.

[0003] Technical Field The present disclosure relates to recombinant nephrosin polypeptides, polynucleotides encoding recombinant nephrosin polypeptides, and methods of expressing recombinant polynucleotides. The use of recombinant nephrosin polypeptides for therapeutic and other purposes is further provided.

Background Art

[0004] Background Celiac disease is a chronic autoimmune disorder that typically affects the small intestine. The symptoms of this disease are caused by a reaction to gluten proteins and other storage proteins in consumed cereal products (e.g., serpins, purinins, etc.). Clinically, this disease can be partially detected by quantifying antibodies specific to gluten and tissue transglutaminase (tTG). The autoimmune response leads to the development of small intestinal mucosal villous atrophy and mucosal inflammation with crypt hyperplasia. The symptoms of celiac disease can vary among individuals and can include one or more of fatigue, chronic diarrhea, constipation, nutrient malabsorption, weight loss, abdominal distension, anemia, and a substantially increased risk of developing osteoporosis and intestinal malignancies (e.g., lymphoma and carcinoma).

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

[0006] Treatment for gluten intolerance, particularly celiac disease, generally involves a strict lifelong gluten-free diet. However, a gluten-free diet is inconvenient and 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 suppressing its role in celiac disease. One such protease is the neprosin class of prolyl endopeptidase (PEP) found in the insectivorous tropical pitcher plants of the Nepenthes species. Neprosin is one of the two major proteins of gluten along with gliadin and can hydrolyze proline-rich gliadin, which is associated with the induction of celiac disease in susceptible individuals. Since the amount of neprosins present in Nepenthes pitcher plants is small, other sources of neprosins are desirable for the evaluation of proteases in therapeutic applications. Summary of the Invention Means for Solving the Problems

[0007] Abstract The present disclosure provides a recombinant neprosin polypeptide, a method for expressing a recombinant neprosin polypeptide that results in efficient expression of the neprosin, particularly in a soluble form, and the use of the recombinant neprosin polypeptide in the treatment of diseases or conditions associated with gluten intolerance such as celiac disease.

[0008] In one aspect, the present disclosure provides a recombinant nephrosin 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 prepropolypeptide, propolypeptide, mature polypeptide, or reference sequence corresponding to the polypeptide of SEQ ID NO: 1, wherein the amino acid sequence comprises substitutions with amino acids other than asparagine (N) at least at amino acid positions 68, 145, 152, or 253 or combinations thereof and their equivalent positions, where the amino acid positions are relative to the reference sequence corresponding to SEQ ID NO: 1.

[0009] In some embodiments, the recombinant nephrosin 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 reference sequence corresponding to residues 356-105 of SEQ ID NO: 8 or to the reference sequence corresponding to SEQ ID NO: 8, wherein the amino acid sequence comprises substitutions with amino acids other than asparagine (N) at least at amino acid positions 44, 121, 128, or 229 or combinations thereof, where the amino acid positions are relative to SEQ ID NO: 8.

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

[0011] In some embodiments, the amino acid sequence of the recombinant nephrosin polypeptide comprises at least a substitution with an amino acid other than asparagine (N) at amino acid position 121. In some embodiments, the amino acid sequence of the recombinant nephrosin polypeptide comprises residues 356-105 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 at least a substitution with an amino acid other than asparagine (N) at amino acid position 128. In some embodiments, the amino acid sequence of the recombinant neprosin polypeptide comprises residues 356-105 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 at least a substitution with an amino acid other than asparagine (N) at amino acid position 229. In some embodiments, the amino acid sequence of the recombinant neprosin polypeptide comprises residues 356-105 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 at least a substitution with an amino acid other than asparagine (N) at amino acid positions 121 and 229. In some embodiments, the amino acid sequence of the recombinant neprosin polypeptide comprises residues 356-105 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 at least a substitution with an amino acid other than asparagine (N) at amino acid positions 44, 128, and 229. In some embodiments, the amino acid sequence of the recombinant neprosin polypeptide comprises residues 356-105 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 at least a substitution with an amino acid other than asparagine (N) at amino acid positions 44, 121, 128, and 229. In some embodiments, the amino acid sequence of the recombinant neprosin polypeptide comprises residues 356-105 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 at least substitutions with amino acids other than asparagine (N) at amino acid positions 44 and 229. In some embodiments, the amino acid sequence of the recombinant neprosin polypeptide comprises residues 356-105 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 has 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 356-105 of SEQ ID NO: 8, or to a reference sequence corresponding to SEQ ID NO: 8, and the amino acid sequence further comprises a signal sequence that is functional in mammalian cells, insect cells, or fungal cells. In some embodiments, the amino acid sequence of the recombinant neprosin polypeptide comprises residues 356-105 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 positions 44, 121, 128 or 229, where the amino acid positions are compared to SEQ ID NO: 8. In some embodiments, the recombinant neprosin polypeptide is glycosylated, but is not glycosylated at least at amino acid positions 44, 121, 128 or 229, where the amino acid positions are compared to SEQ ID NO: 8. In some embodiments, the recombinant neprosin polypeptide is not glycosylated.

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

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

[0023] In another aspect, a host cell comprising the expression vector is further 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 the 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 an Sf9 cell, an Sf21 cell, a Schneider 2 or a High Five cell. In some embodiments, the polynucleotide sequence encoding the recombinant neprosin polypeptide is codon-optimized for expression in insect cells.

[0026] In some embodiments, the host cell comprises a fungal cell comprising an expression vector comprising 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 of the genus 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 of expressing a recombinant neprosin polypeptide, the method comprising culturing a host cell comprising an expression vector comprising a polynucleotide sequence encoding the recombinant neprosin polypeptide under suitable conditions such that the recombinant neprosin polypeptide is expressed.

[0028] In some embodiments of the method, the expressed recombinant nepocin polypeptide is the mature nepocin polypeptide. In some embodiments, the expressed recombinant nepocin polypeptide is the propeptide / proenzyme form of the nepocin polypeptide. In some embodiments, the expressed recombinant nepocin polypeptide is the prepropeptide / preproenzyme form of the nepocin polypeptide. In some embodiments, the expressed recombinant nepocin polypeptide is secreted. In some embodiments, the recombinant nepocin polypeptide is expressed in a soluble form.

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

[0030] In another aspect, the present disclosure provides a method for preparing a mature nepocin polypeptide, particularly under in vitro conditions, the method comprising treating a full-length nepocin polypeptide, or a prepropeptide or propeptide of the nepocin polypeptide, under acidic conditions suitable for the formation of the mature nepocin 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 nepocin polypeptide is prepared by proteolytic cleavage. In some embodiments, the mature nepocin prepared by this method has proteolytic activity.

[0031] In a further 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 present 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 or a pharmaceutical composition thereof as described herein. In some embodiments, the recombinant neprosin polypeptide or a pharmaceutical composition thereof is administered prior to, concurrently with, or subsequent to the consumption of gluten-containing food. In some embodiments, the disease or condition associated with gluten intolerance is celiac disease. BRIEF DESCRIPTION OF THE DRAWINGS

[0033]

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Mode for Carrying Out the Invention

[0043] Detailed Description The present disclosure provides a recombinant neprosin polypeptide, a recombinant polynucleotide encoding the recombinant neprosin polypeptide, a host cell containing the recombinant polynucleotide, and a method for expressing the recombinant neprosin polypeptide using the host cell. The present disclosure further provides a recombinant neprosin polypeptide, particularly prepared from a host cell in a soluble form, for accessing therapeutic efficacy and for use in the treatment of gluten intolerance, and a method for preparing a proteolytically active form of recombinant neprosin. Abbreviations and Definitions

[0044] Unless otherwise defined, all technical and scientific terms used herein generally have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Generally, the nomenclature used herein as well as laboratory procedures in cell culture, molecular genetics, microbiology, organic chemistry, analytical chemistry, and nucleic acid chemistry described below are those well known and commonly employed in the art. Such techniques are well known and described in numerous texts and references well known to those skilled in the art. Standard techniques or modified versions thereof are used in chemical synthesis and chemical analysis.

[0045] Any suitable methods and materials similar to or equivalent to the methods and materials described herein may be found for use in the practice of the present invention, although some methods and materials are described herein. Methodologies, protocols, and reagents may vary depending on the context in which they are used by those of ordinary skill in the art, and it is to be understood that the present invention is not limited to the specific methodologies, protocols, and reagents described. Accordingly, the terms defined immediately below are more fully described by reference to the entire present application. All patents, patent applications, papers, and publications referred to herein, both above and below, are hereby expressly incorporated by reference into this specification.

[0046] As used herein, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise.

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

[0048] It is further to be understood that where an embodiment is described using the term "comprising" and its cognates, this embodiment may also be described using the terms "consisting essentially of" or "consisting of."

[0049] A numerical range includes the numbers defining the range. Thus, any numerical range disclosed herein is intended to encompass any narrower numerical ranges that fall within such broader numerical ranges, as if such narrower numerical ranges were all expressly written herein. It is also intended that any maximum (or minimum) numerical limit disclosed herein includes any lower (or higher) numerical limit, as if such lower (or higher) numerical limits were expressly written herein.

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

[0051] Furthermore, the headings provided herein are not limitations of the various aspects or embodiments of the invention obtained by reference to the entire application. Accordingly, the terms defined immediately below are more fully defined by reference to the entire application. Nevertheless, a number of terms are defined below to facilitate understanding of the invention.

[0052] The "EC" number refers to the enzyme nomenclature of the Nomenclature Committee 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 they catalyze.

[0053] "ATCC" refers to the American Type Culture Collection, the bio-repository collection which 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 denote a polymer of at least two nucleotides, either deoxyribonucleotides or ribonucleotides. The abbreviations used for the gene code nucleosides are conventional and are as follows: adenosine (A); guanosine (G); cytidine (C); thymidine (T); and uridine (U). Unless otherwise specified, the omitted nucleoside may be either a ribonucleoside or a 2'-deoxyribonucleoside. Nucleosides can be specified as either ribonucleosides or 2'-deoxyribonucleosides, either on an individual basis or on an aggregate basis. When a nucleic acid sequence is presented as a series of one-letter abbreviations, the sequence is presented in the 5' to 3' direction according to common convention, and the phosphate is not shown.

[0056] "Double-stranded" and "ds" refer to a double-stranded nucleic acid (e.g., DNA) molecule composed of two single-stranded polynucleotides where the sequences are complementary (A pairs with T, and C pairs with G), arranged in an antiparallel 5' to 3' directionality, 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 herein to denote a polymer of at least two amino acids covalently linked by amide bonds, regardless of length or post-translational modification (e.g., glycosylation or phosphorylation). Unless otherwise indicated, amino acid sequences are written from left to right in the amino to carboxy directionality.

[0058] "Amino acid" is referred to herein by either its generally known three-letter symbol or the one-letter symbol recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Abbreviations used for amino acids encoded by genes 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, the amino acid is of either the L- or D-configuration with respect to the α-carbon (C α ) unless otherwise specified by "L" or "D" preceding the abbreviation or unless it is clear from the context in which the abbreviation is used. For example, "Ala" designates alanine without specifying the configuration with respect to the α-carbon, while "D-Ala" and "L-Ala" designate D-alanine and L-alanine, respectively. When one-letter abbreviations are used, capital letters designate amino acids of the L-configuration with respect to the α-carbon and lowercase letters designate amino acids of the D-configuration with respect to the α-carbon. For example, "A" designates L-alanine and "a" designates D-alanine. When a polypeptide sequence is presented as a series of one-letter or three-letter abbreviations (or a mixture thereof), the sequence is presented in the direction from amino (N) to carboxy (C) according to common convention.

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

[0060] "Neprosin" refers to a prolyl endoprotease first isolated from the pitcher leaf secretion of Nepenthes species. Neprosin is known to cleave proteins at the carboxy terminus with respect to proline with high specificity. The enzyme is active at about pH 2 to about pH 5. Neprosin, as used herein, includes isoforms, isotypes, homologs, orthologs, variants, and recombinant neprosins, 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 the Enzyme Commission number EC3.4.23.12, and includes any isoforms, isotypes, and variants of nepenthesin, such as nepenthesin I and nepenthesin II, nepenthesin isoforms, and recombinant nepenthesin, and salts thereof. Nepenthesin is an aspartic protease of plant origin that can be isolated or concentrated from various plant sources, such as the pitcher leaf secretion of Nepenthes, which is commonly known as the tropical pitcher plant. 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] "Protein", "propolypeptide", or "propeptide" refers to a precursor protein, polypeptide, or peptide that is processed by post-translational modification to form a bioactive protein, polypeptide, or peptide. In some embodiments, the post-translational modification is a cleavage reaction that forms the protein, polypeptide, or peptide. "Proenzyme" refers to a precursor polypeptide that is processed by post-translational modification, particularly a cleavage reaction, to form an active enzyme.

[0064] "Preproprotein", "prepropolypeptide", or "prepropeptide" refers to a precursor protein, polypeptide, or peptide that contains a signal sequence and can be processed by post-translational modification, particularly a cleavage reaction, to generate a protein, propolypeptide, or propeptide. Generally, the cleavage reaction removes the signal sequence to generate the protein, propolypeptide, or propeptide. "Preproenzyme" refers to a precursor protein, polypeptide, or peptide that is processed by post-translational modification, particularly a cleavage reaction, to remove the signal sequence and form a proenzyme.

[0065] "Full-length", in the context of a protein or polypeptide, refers to a protein or polypeptide that has not been processed to alter 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 a cell, polynucleotide, or polypeptide, refer to a material or a material that has been modified in a manner that would not otherwise occur in nature, or that corresponds to the native or natural form of a material but has been produced or obtained from synthetic materials and / or by manipulation using recombinant techniques, where the manipulation has been performed to produce or obtain a material that is the same as the native or natural form of the material.

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

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

[0069] The term "percent (%) sequence identity" is used herein to refer to the comparison between polynucleotides and polypeptides and is determined by comparing two optimally aligned sequences over a comparison window, where the portion of the polynucleotide or polypeptide sequence in the comparison window may include additions or deletions (i.e., gaps) as compared to the reference sequence for optimal alignment of the two sequences. The percentage can be calculated by determining the number of positions at which the identical nucleic acid bases or amino acid residues are present in both sequences, obtaining the number of matched positions, dividing the number of matched positions by the total number of positions in the comparison window, and multiplying the result by 100 to obtain the percentage of sequence identity. Alternatively, the percentage can be calculated by determining the number of positions at which the identical nucleic acid bases or amino acid residues are present in both sequences or by aligning the nucleic acid bases or amino acid residues with gaps to obtain the number of matched positions, dividing the number of matched positions by the total number of positions in the comparison window, and multiplying the result by 100 to obtain the percentage of sequence identity. Those skilled in the art are well aware that there are many established algorithms available for aligning two sequences. Optimal alignment of sequences for comparison can be performed, for example, by the local homology algorithm of Smith and Waterman (Smith and Waterman, Adv. Appl. Math., 1981, 2:482), the homology alignment algorithm of Needleman and Wunsch (Needleman and Wunsch, J. Mol. Biol., 1970, 48:443), the similarity search method of Pearson and Lipman (Pearson and Lipman, Proc. Natl. Acad. Sci. USA, 1988, 85:2444), computerized implementations of these algorithms (e.g., GAP, BESTFIT, FASTA, and TFASTA in the GCG Wisconsin software package), or by visual inspection.Examples of algorithms suitable for determining percent sequence identity and sequence similarity include, but are not limited to, the 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, 3389-3402). Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information website. This algorithm first identifies high-scoring sequence pairs (HSPs) by identifying short words of length "W" in the query sequence that either match or satisfy some positive-valued threshold score "T" when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold (see Altschul et al, supra). These initial neighborhood word hits act as seeds for initiating a search to find longer HSPs that contain them. The word hits are then extended in both directions along each sequence as far as possible while the cumulative alignment score can be increased. For nucleotide sequences, the cumulative score is calculated using the parameters "M" (reward score for a pair of matching residues; always >0) and "N" (penalty score for a mismatching residue; always <0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction is stopped when: the cumulative alignment score falls off by the quantity "X" from its maximum achieved value; the cumulative score goes to zero or below due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses, by default, a word length (W) of 11, an expectation (E) of 10, M = 5, N = -4, and a comparison of both strands.For amino acid sequences, the BLASTP program, by default, uses a word length (W) of 3, an expectation value (E) of 10, and the BLOSUM62 scoring matrix (see, e.g., Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA, 1989, 89:10915). Exemplary determination of sequence alignment and % sequence identity can be made using the BESTFIT or GAP programs in the GCG Wisconsin software package (Accelrys, Madison WI) with the default parameters provided.

[0070] A "reference sequence" refers to a defined sequence used as a basis for sequence comparison. A reference sequence can be a subset of a larger sequence, e.g., a segment of a full-length gene or polypeptide sequence. Generally, a reference sequence is at least 20 nucleotides or amino acid residues in length, at least 25 residues in length, at least 50 residues in length, at least 100 residues in length, or the full length of a nucleic acid or polypeptide. Since two polynucleotides or polypeptides can each (1) contain sequences that are similar between the two sequences (i.e., portions of the complete sequence) and (2) further contain sequences that vary widely between the two sequences, sequence comparison between two (or more) polynucleotides or polypeptides is typically performed by comparing the sequences of the two polynucleotides or polypeptides over a "comparison window" to identify and compare local regions of sequence similarity. In some embodiments, a "reference sequence" can be based on a primary amino acid sequence, and the reference sequence can be a sequence having one or more changes from the primary sequence.

[0071] "Comparison window" refers to a conceptual segment of at least about 20 contiguous nucleotide positions or amino acid residues, the sequences can be compared to a reference sequence of at least 20 contiguous nucleotides or amino acids, and the portion of the sequence in the comparison window can include an addition or deletion (i.e., a gap) of 20 percent or less compared to the reference sequence (excluding additions or deletions) for optimal alignment of the two sequences. The comparison window can be longer than 20 contiguous residues and can include windows of 30, 40, 50, 100 or more residues as needed.

[0072] As used herein, "corresponding to", "with reference to" and "compared to" when used in the context of numbering of a given amino acid or polynucleotide sequence refer to the numbering of residues of the designated reference sequence when the given amino acid or polynucleotide sequence is compared to the reference sequence. In other words, the residue numbers or residue positions of a given polymer are designated with respect to the reference sequence rather than the actual numerical position of the residues within the given amino acid or polynucleotide sequence. For example, a given amino acid sequence, such as the sequence of a recombinant nepocym polypeptide, can be aligned with a reference sequence by introducing gaps to optimize residue matches between the two sequences. In such cases, gaps are present, but the numbering of residues in the given amino acid or polynucleotide sequence is done with respect to the reference sequence to which it is aligned.

[0073] "Amino acid difference" and "residue difference" refer to the difference in amino acid residues at a position in a polypeptide sequence compared to the amino acid residue at the corresponding position in a reference sequence. The position of the amino acid difference is generally referred to herein as "Xn", where n refers to the corresponding position in the reference sequence upon which the residue difference is based. 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 in amino acid residues at the polypeptide position corresponding to position 121 of SEQ ID NO:8. Thus, if the reference polypeptide of SEQ ID NO:8 has asparagine at position 121, "the residue difference at position X121 compared to SEQ ID NO:8" refers to an amino acid substitution by any residue other than asparagine at the polypeptide position corresponding to position 121 of SEQ ID NO:8. In some examples herein, a specific amino acid residue difference at a position is shown as "XnY", where "Xn" designates the corresponding residue and position of the reference polypeptide (as described above), and "Y" is the one-letter identifier of the amino acid found in the engineered or recombinant polypeptide (i.e., a residue different from that in the reference polypeptide). In some embodiments, an amino acid difference, e.g., a substitution, is denoted by the abbreviation "nY" without an identifier of the residue in the reference sequence.

[0074] "Conservative amino acid substitution" refers to the substitution of one residue with a different residue having a similar side chain, and thus typically involves the substitution of an amino acid in a polypeptide with an amino acid within the same or a similar defined class of amino acids. By way of example and not limitation, an amino acid having an aliphatic side chain can be substituted with another aliphatic amino acid (e.g., alanine, valine, leucine, and isoleucine); an amino acid having a hydroxyl side chain is substituted with another amino acid having a hydroxyl side chain (e.g., serine and threonine); an amino acid having an aromatic side chain is substituted with another amino acid having an aromatic side chain (e.g., phenylalanine, tyrosine, tryptophan, and histidine); an amino acid having a basic side chain is substituted with another amino acid having a basic side chain (e.g., lysine and arginine); an amino acid having an acidic side chain is substituted with another amino acid having an acidic side chain (e.g., aspartic acid or glutamic acid); and / or a hydrophobic or hydrophilic amino acid is replaced with another hydrophobic or hydrophilic amino acid, respectively.

[0075] "Non-conservative substitution" refers to the substitution of an amino acid in a polypeptide with an amino acid having significantly different side chain characteristics. Non-conservative substitutions can use amino acids from outside rather than within defined groups and can affect (a) the structure of the peptide backbone in the region of substitution (e.g., proline substituted for glycine); (b) charge or hydrophobicity; or (c) the bulk of the side chain. By way of example and not limitation, exemplary non-conservative substitutions can be acidic amino acids substituted with basic or aliphatic amino acids; aromatic amino acids substituted with small amino acids; and hydrophilic amino acids substituted with hydrophobic amino acids.

[0076] "Deletion" refers to a modification to a polypeptide by the removal of one or more amino acids from a reference polypeptide. Deletions can include the removal of one or more amino acids, two or more amino acids, five or more amino acids, ten or more amino acids, fifteen or more amino acids, or twenty or more amino acids, up to 10% of the total number of amino acids constituting the reference enzyme, or up to 20% of the total number of amino acids, while retaining the enzymatic activity and / or properties of the neprosin polypeptide. Deletions can be directed to the internal portion and / or the terminal portions of the polypeptide. In various embodiments, deletions can include contiguous segments or can be non-contiguous.

[0077] "Insertion" refers to a modification to a polypeptide by the addition of one or more amino acids to a reference polypeptide. Insertions can be made into the internal portion of the polypeptide or at the carboxy or amino terminus. Insertions, as used herein, include fusion proteins, as is known in the art. Insertions can be adjacent segments of amino acids or can be separated by one or more of the amino acids in a naturally occurring polypeptide.

[0078] "Functional fragment" and "bioactive fragment" are used interchangeably herein to refer to a polypeptide that has amino-terminal and / or carboxy-terminal deletion(s) and / or internal deletions, but in which the remaining amino acid sequence is identical to the corresponding positions in the sequence to which it is being compared (e.g., a 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 are naturally associated with it. This term encompasses polypeptides that have been removed or purified from their natural environment or expression system (e.g., host cell or in vitro synthesis). The recombinant neprosin polypeptide may be present intracellularly, in the cell culture medium, or prepared in various forms such as lysates or isolated preparations. Thus, 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., is more abundant than any other individual macromolecular species in the composition on a molar or weight basis), and generally, a composition is substantially purified when the species of interest constitutes at least about 50 percent of the macromolecular species present, on a molar or % weight basis. Generally, a substantially pure neprosin polypeptide composition constitutes 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 until it is essentially homogeneous (i.e., contaminant species cannot be detected in the composition by conventional detection methods), and the composition consists essentially of a single macromolecular species. Solvent species, small molecules (<500 daltons), and elemental ion species are not considered macromolecular species. In some embodiments, the isolated recombinant neprosin polypeptide polypeptide is a substantially pure polypeptide composition.

[0081] "Proteolytic activity" and "proteolysis" are used interchangeably herein to refer to the breakdown of a protein into smaller polypeptides or amino acids. The breakdown of a protein is generally the result of hydrolysis of peptide bonds by protease (proteinase) enzymes. Exemplary protease enzymes include, but are not limited to, neprosin, nepenthesin, pepsin, trypsin, chymotrypsin, elastase; carboxypeptidase A and B, and peptidases (e.g., aminopeptidase, dipeptidase, and enteropeptidase).

[0082] "Codon-optimized" refers to a change in the codons of a polynucleotide encoding a protein to codons that are preferentially used in a particular organism such that the encoded protein is expressed more efficiently in that organism. The genetic code is degenerate in that most amino acids are represented by several codons, called "synonymous" or "synonymous" codons, but codon usage by a particular organism is non-random and biased towards certain codon triplets. This codon usage bias can be higher when referring to a given gene, genes of common function or ancestral origin, highly expressed proteins versus low copy number proteins, and the collective protein coding regions of an organism's genome. In some embodiments, the polynucleotide encoding the neprosin polypeptide is codon-optimized for optimal production from the host organism selected for expression.

[0083] As used herein, "control sequences" refers to all components necessary or advantageous for the expression of a polynucleotide and / or polypeptide of the present disclosure. Each control sequence may be native or foreign to the nucleic acid sequence encoding the polypeptide. Such control sequences include, but are not limited to, leader, polyadenylation sequences, propeptide sequences, promoter sequences, signal peptide sequences, start sequences and transcription terminators. At a minimum, the control sequences include a promoter, as well as transcription and translation termination signals. In some embodiments, linkers are provided in the control sequences for the purpose of introducing specific restriction sites that facilitate ligation of the coding region of the nucleic acid sequence encoding the polypeptide and the control sequences.

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

[0085] "Heterologous" or "recombinant" refers to the relationship between two or more nucleic acid or polypeptide sequences (e.g., promoter sequences, signal peptides, terminator sequences, etc.) that are derived from different sources and are not associated in nature.

[0086] "Promoter sequence" refers to a nucleic acid sequence recognized by a host cell for the expression of a polynucleotide of interest, such as a coding sequence. The promoter sequence contains transcriptional control sequences that mediate the expression of the polynucleotide of interest. A 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 or intracellular polypeptide that is either homologous or heterologous to the host cell.

[0087] A "vector" is a DNA construct for introducing a polynucleotide sequence into a cell. In some embodiments, the vector is an expression vector operably linked to suitable control sequences that can effect the expression of a polypeptide encoded by the 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, also includes a transcription terminator sequence.

[0088] The term "expression" includes 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 encompasses the secretion of a polypeptide from a cell.

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

[0090] "Producing" refers to the production of proteins and / or other compounds by a cell. This 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 encompasses the secretion of a polypeptide from a cell.

[0091] "Host cell" and "host strain" refer to a suitable host for an expression vector containing 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 transformed or transfected with a vector constructed using recombinant DNA techniques known in the art.

[0092] "Effective amount" means an amount sufficient to produce a desired result. One of ordinary skill in the art can determine an effective amount by using routine experimentation.

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

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

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

[0096] "Pharmaceutically acceptable" means a material that can be administered to a subject without causing any undesirable biological effects and without interacting in a harmful manner with any of the components contained therein and possessing the desired biological activity.

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

[0098] "Carrier", when used in reference to a pharmaceutical composition, means any of a standard pharmaceutical carrier, buffer, and excipient such as a stabilizer, preservative, and adjuvant.

[0099] "Therapeutically effective amount", when used with reference to the symptoms of a disease / condition, refers to the amount and / or concentration of a compound (e.g., a recombinant nepocym polypeptide) that alleviates, attenuates, or eliminates one or more symptoms of the disease / condition, or prevents or delays the onset of the symptom(s). "Therapeutically effective amount", when used with reference to a disease / condition, refers to the amount and / or concentration of a composition (e.g., a recombinant nepocym polypeptide) that alleviates, attenuates, or eliminates the disease / condition. In some embodiments, the term is used with reference to the amount of a composition that induces a biological (e.g., medical) response in a tissue, system, or animal subject being investigated by a researcher, physician, veterinarian, or other clinician.

[0100] "Treating" or "treatment" of a disease, disorder, or syndrome, as used herein, includes (i) preventing the disease, disorder, or syndrome from occurring in a subject, i.e., not causing the clinical symptoms of the disease, disorder, or syndrome to develop in an animal that may be exposed to or is predisposed to the disease, disorder, or syndrome but has not yet experienced or manifested the symptoms of the disease, disorder, or syndrome; (ii) inhibiting the disease, disorder, or syndrome, i.e., arresting its development; and (iii) alleviating the disease, disorder, or syndrome, i.e., causing regression of the disease, disorder, or syndrome. Accordingly, the terms "treating", "treat", and "treatment" encompass both prophylactic (e.g., preventive) and symptomatic treatment. As is known in the art, adjustments may be necessary for systemic versus localized delivery, age, body weight, general health, sex, diet, time of administration, drug interactions, and the severity of the condition, and will be ascertainable by one of ordinary skill in the art.

[0101] "Concurrent administration" or "co-treatment", as used herein, includes the administration of agents together, or before or after one another.

[0102] "Modulate," "attenuate," or "alleviate" means treating any disease or disorder in a subject, such as a mammal, including preventing or protecting from a disease or disorder, e.g., not causing an abnormal biological response or symptom; inhibiting a disease or disorder, suppressing or inhibiting the onset of an abnormal biological response and / or clinical symptom; and / or alleviating a disease or disorder, e.g., causing regression of an abnormal biological response and / or symptom.

[0103] "Prevent" or "inhibit" refers to prophylactic treatment of a subject in need thereof. Prophylactic treatment can be achieved by providing a subject at risk of developing a disease with a therapeutically effective amount of a therapeutic agent, thereby substantially preventing the onset of the disease.

[0104] "Antigenic food or protein," as used herein, refers to a food containing a protein that can cause an immune and / or inflammatory response in the intestine of a sensitive individual. In a preferred embodiment, the individual is human and the food is intended for human consumption. Antigenic foods include, by way of example and not limitation, wheat, rye, barley, peanuts, nuts and seeds. In some embodiments, the antigenic proteins from these foods include prolamin proteins, 2S albumins, nonspecific lipid transfer proteins, bifunctional α-amylase / protease inhibitors, soybean hydrophobic proteins, indolins, gluten, serpins, purinins, alpha-amylase / protease inhibitors, globulins 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 a wheat protein.

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

[0106] "Gluten sensitivity and related conditions" refers to any condition resulting from intolerance or sensitivity to gluten proteins or peptides. Such conditions include, without limitation, celiac sprue (celiac disease), wheat allergy, gluten sensitivity, gluten-sensitive enteropathy, idiopathic gluten sensitivity and dermatitis herpetiformis. Related conditions include, without limitation, autism, attention deficit hyperactivity disorder (ADHD), rheumatoid arthritis, fibromyalgia, Crohn's disease, nutrient malabsorption and irritable bowel syndrome (IBS).

[0107] "Symptoms of celiac disease" refers to either the symptoms or clinical manifestations of celiac disease. Such symptoms include, without limitation, intestinal inflammation, "foggy mind", depression, anxiety, ADHD-like behavior, abdominal pain, abdominal distension, diarrhea, constipation, headache, migraine, bone or joint pain, chronic fatigue, small intestine damage, the occurrence of tissue transglutaminase (tTG) antibodies, severe acne, vomiting, weight loss, irritability, iron deficiency anemia, arthritis, pins and needles sensations in the limbs, infertility, and stomatitis. The symptoms further include intestinal mucosal villous atrophy with crypt hyperplasia, intestinal mucosal inflammation, malabsorption of nutrients, abdominal distension, as well as a substantially increased risk of developing osteoporosis and intestinal malignancies (lymphoma and carcinoma).

[0108] Recombinant neprosin polypeptides and expressed neprosin polypeptides The pitcher fluid secretions of Nepenthes, an insectivorous pitcher plant commonly known as the tropical pitcher plant, contain several different proteases. Concentrated Nepenthes pitcher fluid has high specificity for gluten peptides rich in proline and glutamine. U.S. Patent Application Publication Nos. 2014 / 0186330 and 2014 / 0140980, which are hereby incorporated by reference in their entirety, describe the activity and specificity of concentrated Nepenthes pitcher fluid and recombinant Nepenthes enzymes. The pitcher fluid is acidic, and the enzymes therein are generally most active at acidic pH values.

[0109] Prolyl endoprotease neprosin, one of the proteases present in the secretions of the pouch leaf, has high proteolytic activity for cleaving proline-rich proteins and oligopeptides such as gluten proteins. The amino acid sequence (SEQ ID NO: 1) of the neprosin polypeptide present in N. x ventrata is shown in Figure 1A. The neprosin polypeptide contains a signal sequence, a prodomain region, and a neprosin protease region. As disclosed herein, analysis of the pH-activated, mature, proteolytically active neprosin polypeptide by N-terminal sequencing indicates that the primary form of the mature, proteolytically active neprosin polypeptide generated at acidic pH starts from the serine residue at amino acid position 129, such that the propeptide is cleaved at acidic pH between the proline and serine residues at amino acid positions 128 and 129 of SEQ ID NO: 1. Without being bound by any theory of the mechanism, N-glycosylation sites are shown at amino acid positions 68, 145, 152, and 253 with reference to SEQ ID NO: 1 in Figure 1A. The propeptide form (SEQ ID NO: 8) of the neprosin polypeptide is provided in Figure 1B, and the prodomain (propeptide) and protease region (neprosin 1) are indicated by block 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, particularly active at low pH (e.g., about 3 - 5), and cleaves peptides on the carboxy (C) - terminal side of proline. This cleavage appears to be highly specific. The activity and specificity of neprosin in cleaving gluten proteins into peptides with reduced immunogenicity provide a basis for treating celiac disease and other inflammatory disorders related to the immunogenicity of gluten proteins. Accordingly, the present disclosure provides recombinant neprosin polypeptides, and in particular, methods for expressing recombinant neprosin polypeptides in a soluble form. In some embodiments, the recombinant neprosin polypeptide is in a pro - polypeptide form. In some embodiments, the pro - polypeptide form can be cleaved under acidic pH or by proteases, etc., to form a mature polypeptide.

[0111] In some embodiments, the 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 pro - polypeptide of the recombinant neprosin polypeptide has 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 the choice of host cell for the expression of the recombinant neprosin polypeptide, e.g., mammalian cells, insect cells, or fungal cells.

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

[0113] In some embodiments, the recombinant nepocin polypeptide is not glycosylated. In some embodiments, the prepropolypeptide of recombinant nepocin is not glycosylated. In some embodiments, the propolypeptide of recombinant nepocin is not glycosylated. In some embodiments, the mature polypeptide of recombinant nepocin is not glycosylated.

[0114] In one aspect, the present 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 the prepropolypeptide, propolypeptide, mature polypeptide, or reference sequence corresponding to the polypeptide of SEQ ID NO: 1, wherein the amino acid sequence comprises substitutions with amino acids other than asparagine (N) at least at amino acid positions 68, 145, 152, or 253 or combinations thereof and their equivalent positions, where the amino acid positions are 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 the prepropolypeptide, propolypeptide, mature polypeptide, or reference sequence corresponding to the polypeptide of SEQ ID NO: 1, wherein the amino acid sequence comprises substitutions with amino acids other than asparagine (N) at least at amino acid position 68 or its equivalent position, where 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 or its combination 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 the prepropolypeptide, propolypeptide, mature polypeptide, or reference sequence corresponding to the polypeptide of SEQ ID NO:1, and the amino acid sequence comprises a substitution with an amino acid other than asparagine (N) at least at amino acid position 145, or its equivalent position, where the amino acid position is compared 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 or its combination 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 the prepropolypeptide, propolypeptide, mature polypeptide, or reference sequence corresponding to the polypeptide of SEQ ID NO:1, and the amino acid sequence comprises a substitution with an amino acid other than asparagine (N) at least at amino acid position 152, or its equivalent position, where the amino acid position is compared 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 or its combination is A.

[0118] In some embodiments, the recombinant nepocin 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 prepropolypeptide, propolypeptide, mature polypeptide, or reference sequence corresponding to the polypeptide of SEQ ID NO: 1, the amino acid sequence comprising a substitution with an amino acid other than asparagine (N) at least at amino acid position 253, or its equivalent position, where 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 or its combination is A.

[0119] In some embodiments, the recombinant nepocin 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 prepropolypeptide, propolypeptide, mature polypeptide, or reference sequence corresponding to the polypeptide of SEQ ID NO: 1, the amino acid sequence comprising a substitution with an amino acid other than asparagine (N) at least at amino acid positions 68 and 145, or their equivalent positions, where the amino acid position is 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 or their combination 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 the prepropolypeptide, propolypeptide, mature polypeptide, or reference sequence corresponding to the polypeptide of SEQ ID NO: 1, the amino acid sequence comprising substitutions with amino acids other than asparagine (N) at least at amino acid positions 68 and 152, or their equivalent positions, where 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 position 68 and 152 or a combination thereof 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 the prepropolypeptide, propolypeptide, mature polypeptide, or reference sequence corresponding to the polypeptide of SEQ ID NO: 1, the amino acid sequence comprising substitutions with amino acids other than asparagine (N) at least at amino acid positions 68 and 253, or their equivalent positions, where 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 position 68 and 253 or a combination thereof is A.

[0122] In some embodiments, the recombinant nepocin 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 prepropolypeptide, propolypeptide, mature polypeptide, or reference sequence corresponding to the polypeptide of SEQ ID NO: 1, the amino acid sequence comprising substitutions with amino acids other than asparagine (N) at least at amino acid positions 145 and 152, or their equivalent positions, where 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 or a combination thereof is A.

[0123] In some embodiments, the recombinant nepocin 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 prepropolypeptide, propolypeptide, mature polypeptide, or reference sequence corresponding to the polypeptide of SEQ ID NO: 1, the amino acid sequence comprising substitutions with amino acids other than asparagine (N) at least at amino acid positions 145 and 253, or their equivalent positions, where 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 or a combination thereof is A.

[0124] In some embodiments, the recombinant nepocin 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 prepropolypeptide, propolypeptide, mature polypeptide, or reference sequence corresponding to the polypeptide of SEQ ID NO: 1, the amino acid sequence comprising substitutions with amino acids other than asparagine (N) at least at amino acid positions 152 and 253, or their equivalent positions, where 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 position 152 and 253 or a combination thereof is A.

[0125] In some embodiments, the recombinant nepocin 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 prepropolypeptide, propolypeptide, mature polypeptide, or reference sequence corresponding to the polypeptide of SEQ ID NO: 1, the amino acid sequence comprising substitutions with amino acids other than asparagine (N) at least at amino acid positions 68, 145 and 152, or their equivalent positions, where 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 position 68, 145 and 152 or a combination thereof is A.

[0126] In some embodiments, the recombinant nepocin 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 prepropolypeptide, propolypeptide, mature polypeptide, or reference sequence corresponding to the polypeptide of SEQ ID NO: 1, the amino acid sequence comprising substitutions with amino acids other than asparagine (N) at least at amino acid positions 68, 145, and 253, or their equivalent positions, where 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 position 68, 145, and 253 or a combination thereof is A.

[0127] In some embodiments, the recombinant nepocin 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 prepropolypeptide, propolypeptide, mature polypeptide, or reference sequence corresponding to the polypeptide of SEQ ID NO: 1, the amino acid sequence comprising substitutions with amino acids other than asparagine (N) at least at amino acid positions 68, 152, and 253, or their equivalent positions, where 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 position 68, 152, and 253 or a combination thereof 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 the prepropolypeptide, propolypeptide, mature polypeptide, or reference sequence corresponding to the polypeptide of SEQ ID NO: 1, the amino acid sequence comprising substitutions by amino acids other than asparagine (N) at least at amino acid positions 145, 152, and 253, or their equivalent positions, where 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 position 145, 152, and 253 or a combination thereof 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 the prepropolypeptide, propolypeptide, mature polypeptide, or reference sequence corresponding to the polypeptide of SEQ ID NO: 1, the amino acid sequence comprising substitutions by amino acids other than asparagine (N) at least at amino acid positions 68, 145, 152, and 253, or their equivalent positions, where 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 position 68, 145, 152, and 253 or a combination thereof 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 the prepropolypeptide, propolypeptide, mature polypeptide, or reference sequence corresponding to the polypeptide of SEQ ID NO:1, the amino acid sequence comprising at least substitution N68A, N145A, N152A, or N253A, or their equivalent positions, and combinations thereof, where 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 the prepropolypeptide, propolypeptide, mature polypeptide, or reference sequence corresponding to the polypeptide of SEQ ID NO:1, the amino acid sequence comprising at least substitution N68A, or its equivalent position.

[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 the prepropolypeptide, propolypeptide, mature polypeptide, or reference sequence corresponding to the polypeptide of SEQ ID NO:1, the amino acid sequence comprising at least substitution N145A, or its equivalent position.

[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 the prepropolypeptide, propolypeptide, mature polypeptide, or reference sequence corresponding to the polypeptide of SEQ ID NO: 1, and the amino acid sequence comprises at least substitution N152A, or its equivalent position.

[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 the prepropolypeptide, propolypeptide, mature polypeptide, or reference sequence corresponding to the polypeptide of SEQ ID NO: 1, and the amino acid sequence comprises at least substitution N253A, or their equivalent positions.

[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 the prepropolypeptide, propolypeptide, mature polypeptide, or reference sequence corresponding to the polypeptide of SEQ ID NO: 1, and the amino acid sequence comprises at least substitutions N68A and N152A, or their equivalent positions.

[0136] In some embodiments, the recombinant nepocin 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 prepropolypeptide, propolypeptide, mature polypeptide, or reference sequence corresponding to the polypeptide of SEQ ID NO: 1, and the amino acid sequence comprises at least the substitutions N68A and N253A, or their equivalent positions.

[0137] In some embodiments, the recombinant nepocin 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 prepropolypeptide, propolypeptide, mature polypeptide, or reference sequence corresponding to the polypeptide of SEQ ID NO: 1, and the amino acid sequence comprises at least the substitutions N145A and N253A, or their equivalent positions.

[0138] In some embodiments, the recombinant nepocin 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 prepropolypeptide, propolypeptide, mature polypeptide, or reference sequence corresponding to the polypeptide of SEQ ID NO: 1, and the amino acid sequence comprises at least the substitutions N152A and N253A, or their equivalent positions.

[0139] In some embodiments, the recombinant nepocin 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 prepolypeptide, propolypeptide, mature polypeptide, or reference sequence corresponding to the polypeptide of SEQ ID NO:1, and the amino acid sequence comprises at least the substitutions N68A, N152A, and N253A, or their equivalent positions.

[0140] In some embodiments, the recombinant nepocin 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 prepolypeptide, propolypeptide, mature polypeptide, or reference sequence corresponding to the polypeptide of SEQ ID NO:1, and the amino acid sequence comprises at least the substitutions N145A, N152A, and N253A, or their equivalent positions.

[0141] In some embodiments, the recombinant nepocin 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 prepolypeptide, propolypeptide, mature polypeptide, or reference sequence corresponding to the polypeptide of SEQ ID NO:1, and the amino acid sequence comprises at least the substitutions N68A, N145A, N152A, and N253A, or their equivalent positions.

[0142] In some embodiments, the recombinant nepocin polypeptide has 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 an amino acid sequence corresponding to residues 381-26 of SEQ ID NO: 44, or a polypeptide sequence comprising its pro-polypeptide or mature polypeptide, and the amino acids at positions 68, 152, and / or 253 are other than asparagine (N).

[0143] In some embodiments, the recombinant nepocin polypeptide has 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 381-26 of SEQ ID NO: 46, or a polypeptide sequence comprising its pro-polypeptide or mature polypeptide, and the amino acid at position 68 is other than asparagine (N).

[0144] In some embodiments, the recombinant nepocin polypeptide has 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 381-26 of SEQ ID NO: 48, or a polypeptide sequence comprising its pro-polypeptide or mature polypeptide, and the amino acid at position 152 is other than asparagine (N).

[0145] In some embodiments, the recombinant nepocin 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 the polypeptide sequence comprising residues 381-26 of SEQ ID NO: 50, or its pro-polypeptide or mature polypeptide, and the amino acid at position 253 is other than asparagine (N).

[0146] In some embodiments, the recombinant nepocin 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 the polypeptide sequence comprising residues 381-26 of SEQ ID NO: 52, or its pro-polypeptide or mature polypeptide, and the amino acids at positions 68 and 253 are other than asparagine (N). In some embodiments, the amino acids at positions 68 and 253 are alanine (A).

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

[0148] In some embodiments, the recombinant neprosin polypeptide has 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 356-105 of SEQ ID NO: 8 or to a reference sequence corresponding to SEQ ID NO: 8, and the amino acid sequence comprises substitutions with amino acids other than asparagine (N) at least at amino acid positions 44, 121, 128, or 229 or combinations thereof, where 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 combinations 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 combinations thereof is A.

[0149] In some embodiments, the recombinant nepocin polypeptide comprises an amino acid sequence comprising residues 356-105 of SEQ ID NO: 8, the amino acid sequence comprising substitutions with amino acids other than asparagine (N) at at least amino acid positions 121, 128, or 229 or combinations thereof, wherein the amino acid positions are relative to SEQ ID NO: 8. In some embodiments, the recombinant nepocin polypeptide comprises an amino acid sequence comprising SEQ ID NO: 8, the amino acid sequence comprising substitutions with amino acids other than asparagine (N) at at least amino acid positions 44, 121, 128, or 229 or combinations 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 combinations 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 combinations thereof is A.

[0150] In some embodiments, the recombinant nepocin 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 SEQ ID NO: 8, the amino acid sequence comprising substitutions with amino acids 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 or combinations thereof is A.

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

[0152] In some embodiments, the recombinant nepocin 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 356-105 of SEQ ID NO: 8 or to a reference sequence corresponding to SEQ ID NO: 8, the amino acid sequence comprising a substitution at at least amino acid position 121 with an amino acid other than asparagine (N), where 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 or a combination thereof is A.

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

[0154] In some embodiments, the recombinant nepocin polypeptide has 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 356-105 of SEQ ID NO: 8, or to a reference sequence corresponding to SEQ ID NO: 8, and the amino acid sequence includes a substitution by an amino acid other than asparagine (N) at least at amino acid position 128, where 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 or a combination thereof is A.

[0155] In some embodiments, the recombinant nepocin polypeptide includes an acid-activated proteolytic activity polypeptide derived from or including a subsequence of SEQ ID NO: 14. In some embodiments, the recombinant nepocin polypeptide includes an amino acid sequence including residues 356-105 of SEQ ID NO: 14. In some embodiments, the recombinant nepocin polypeptide includes an amino acid sequence including SEQ ID NO: 14. In some embodiments, the amino acid sequence including residues 356-105 of SEQ ID NO: 14, or the recombinant nepocin polypeptide including SEQ ID NO: 14, is glycosylated.

[0156] In some embodiments, the recombinant neprosin polypeptide has 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 356-105 of SEQ ID NO: 8, or to a reference sequence corresponding to SEQ ID NO: 8, and the amino acid sequence includes a substitution with an amino acid other than asparagine (N) at least at amino acid position 229, where 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 or a combination thereof is A.

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

[0158] In some embodiments, the recombinant nepocin polypeptide has 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 356 - 105 of SEQ ID NO: 8, or to a reference sequence corresponding to SEQ ID NO: 8, and the amino acid sequence includes substitutions with amino acids other than asparagine (N) at least at amino acid positions 121 and 229, where 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 position 121 and 229 or a combination thereof is A.

[0159] In some embodiments, the recombinant nepocin polypeptide includes an acid-activated proteolytic activity polypeptide derived from SEQ ID NO: 18 or including a subsequence thereof. In some embodiments, the recombinant nepocin polypeptide includes an amino acid sequence including residues 356 - 105 of SEQ ID NO: 18. In some embodiments, the recombinant nepocin polypeptide includes an amino acid sequence including SEQ ID NO: 18. In some embodiments, the amino acid sequence including residues 356 - 105 of SEQ ID NO: 18, or the recombinant nepocin polypeptide including SEQ ID NO: 18, is glycosylated.

[0160] In some embodiments, the recombinant neprosin polypeptide has 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 356 - 105 of SEQ ID NO: 8, or to a reference sequence corresponding to SEQ ID NO: 8, and the amino acid sequence includes substitutions by amino acids other than asparagine (N) at least at amino acid positions 44, 128, and 229, or their equivalent positions, where 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 position 44, 128, and 229, or a combination thereof, is A.

[0161] In some embodiments, the recombinant neprosin polypeptide includes an acid-activated proteolytic activity polypeptide derived from or including a subsequence of SEQ ID NO: 20. In some embodiments, the recombinant neprosin polypeptide includes an amino acid sequence including residues 356 - 105 of SEQ ID NO: 20. In some embodiments, the recombinant neprosin polypeptide includes an amino acid sequence including SEQ ID NO: 20. In some embodiments, the amino acid sequence including residues 356 - 105 of SEQ ID NO: 20, or the recombinant neprosin polypeptide including SEQ ID NO: 20, is glycosylated.

[0162] In some embodiments, the recombinant nepocin polypeptide has 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 356-105 of SEQ ID NO: 8, or to a reference sequence corresponding to SEQ ID NO: 8, and the amino acid sequence includes substitutions by amino acids other than asparagine (N) at least at amino acid positions 44, 121, 128, and 229, where 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 position 44, 121, 128, and 229 or a combination thereof is A.

[0163] In some embodiments, the recombinant nepocin polypeptide includes an acid-activated proteolytic activity polypeptide derived from or including a subsequence of SEQ ID NO: 22. In some embodiments, the recombinant nepocin polypeptide includes an amino acid sequence including residues 356-105 of SEQ ID NO: 22. In some embodiments, the recombinant nepocin polypeptide includes an amino acid sequence including SEQ ID NO: 22. In some embodiments, the amino acid sequence including residues 356-105 of SEQ ID NO: 22, or the recombinant nepocin polypeptide including SEQ ID NO: 22, is not glycosylated.

[0164] In some embodiments, the recombinant nepocin 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 356-105 of SEQ ID NO: 8, or to a reference sequence corresponding to SEQ ID NO: 8, wherein the amino acid sequence comprises substitutions by amino acids other than asparagine (N) at least at amino acid positions 44 and 229, where 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 position 44 and 229 or a combination thereof is A.

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

[0166] In some embodiments, the recombinant nepocin 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 356-105 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 proteolytic activity polypeptide derived from or comprising a subsequence of SEQ ID NO: 8. In some embodiments, the recombinant neprosin polypeptide comprises an amino acid sequence comprising residues 356-105 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 amino acid sequence comprising residues 356-105 of SEQ ID NO: 8, or the recombinant neprosin polypeptide comprising 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 positions 44, 121, 128, or 229, where the amino acid positions are relative to SEQ ID NO: 8. In some embodiments, the recombinant neprosin polypeptide is glycosylated but not glycosylated at least at amino acid positions 44, 121, 128, or 229, where 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 451-96 of SEQ ID NO: 58, 60, 62, 64, 66, 68, or 70. In some embodiments, the recombinant neprosin polypeptide comprises a pro-polypeptide of an amino acid sequence comprising residues 451-96 of SEQ ID NO: 58, 60, 62, 64, 66, 68, or 70. In some embodiments, the recombinant neprosin polypeptide comprises a mature polypeptide of an amino acid sequence comprising residues 451-96 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 signal peptide. In some embodiments, the signal sequence or signal peptide is functional in a host cell that is being or will be used for the expression of the recombinant neprosin polypeptide. In some embodiments, the signal sequence or signal peptide is fused to the pro-polypeptide form of the recombinant neprosin, for example, to form a pre-pro-polypeptide. In some embodiments, the signal sequence or signal peptide is fused to a polypeptide comprising the mature, active form of the recombinant neprosin. In some embodiments, the signal sequence can 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 mammalian or insect cells. Exemplary mammalian or insect signal sequences or signal peptides include, among others, 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, e.g., MGWSCIILFLVATATGVHS (SEQ ID NO: 81), BM40, e.g., MRAWIFFLLCLAGRALA (SEQ ID NO: 82); Secrecon, e.g., MWWRLWWLLLLLLLLWPMVWA (SEQ ID NO: 83); human IgKVIII, e.g., MDMRVPAQLLGLLLLWLRGARC (SEQ ID NO: 84); CD33, e.g., MPLLLLLPLLWAGALA (SEQ ID NO: 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); silkworm fibroin LC, e.g., MKPIFLVLLVVTSAYA (SEQ ID NO: 94); and the honeybee melittin signal peptide of gp64 or gp67, e.g., MLLVNQSHQGFNKEHTSKMVSAIVLYVLLAAAAHSAFA (SEQ ID NO: 95). 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 functional in fungal cells. Exemplary fungal signal sequences or signal peptides include, among others, Pichia pastoris Ost1, e.g., MRFPSIFTAVLFAASSALAAPVNTTTEDETAQIPAEAVIGYLDLEGDFDVAVLPFSNSTNNGLLFINTTIASIAAKEEGVSLDKREAEA (SEQ ID NO: 97); Pichia pastoris Pst1, MRFPSIFTAVLFAASSALAAPANTTTFDETAQIPAFAVIDYSDLFGDFDAAALPLSNSTNNGISSTNTTIASIAAKEFGVLEKRFAFA (SEQ ID NO: 98); S. cerevisiae α - mating factor, e.g., MRFPSIFTAVLFAASSALA (SEQ ID NO: 99); S. cerevisiae invertase, e.g., MLLQAFLFLLAGF (SEQ ID NO: 100); Komagataella pastoris yeast α - factor, e.g., MKPLILSAILITLCITSIAVSAPVEGIFADLHDSSHPYITDDVGVRMSAIKEENPDRHFVGDEIPDEAVIQSFKSKRREILFLLDDIGSAVEFIGSNLAQIEANHLSERVQFSWTHIRKNQPFGKREAEA (SEQ ID NO: 101); S. cerevisiae CYP, e.g., MKAFTSLLCGLGLSTTLAKA (SEQ ID NO: 102); Pichia pastoris PH08, e.g., MDSEPLLPNPNDSRKPANWRRIIKYISLTLAWIGIFSYVYIYHGTA (SEQ ID NO: 103); S. cerevisiae PEP4, e.g., MFSLKALLPLALLLVSANQVAA (SEQ ID NO: 104); S.It includes Saccharomyces cerevisiae SUC2, for example, MLLQAFLFLLAGFAAKISA (SEQ ID NO: 105); Pichia pastoris KAR2, for example, MLSLKPSWLTLAALMYAMLLVVVPFAKPVRA (SEQ ID NO: 106); Pichia pastoris DSE4, for example, MSFSSNVPQLFLLLVLLTNIVSG (SEQ ID NO: 107); Pichia pastoris EXG1, for example, MNLYLITLLFASLCSA (SEQ ID NO: 108); and Pichia pastoris SCW10, for example, MRFSNFLTVSALLTGALG (SEQ ID NO: 109). In some embodiments, the signal sequence or signal peptide includes the yeast α - mating factor prepro sequence comprising residues 1 - 89 of SEQ ID NO: 54.

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

[0174] In some embodiments, an 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. By way of non-limiting example, the signal sequence of SEQ ID NO: 1 can be replaced with an alternative sequence such as a signal sequence for expression in mammalian or insect cells, or a signal sequence for expression in fungal cells such as Pichia or Saccharomyces. In some embodiments, the signal sequence is operably fused to the pro-polypeptide of the recombinant neprosin described herein or, in some embodiments, the mature polypeptide, to facilitate expression and / or processing to prepare the recombinant neprosin polypeptide. Exemplary recombinant neprosin polypeptides having a heterologous signal sequence or signal peptide 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 polypeptides described herein can be fused to various polypeptide sequences, by way of non-limiting example, such as 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 or 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, the fusion is made to the amino (N) terminus of the recombinant neprosin polypeptide. In some embodiments, the fusion is made to the carboxy (C) terminus of the recombinant neprosin polypeptide. In some embodiments, the fusion polypeptide is inserted after the signal sequence and before the neprosin polypeptide to enable expression and secretion of a polypeptide comprising the fusion polypeptide (e.g., a polypeptide tag) and the recombinant neprosin polypeptide.

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

[0177] In some embodiments, the recombinant nepenthesin polypeptide is a nepenthesin derived from the genus Nepenthes, including but not limited to N. x ventrata, N. ampullaria, N. rafflesiana and N. alata.

[0178] In some embodiments, the recombinant nepenthesin 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, 2, 3, 4, 5, or 6.

[0179] In some embodiments, the recombinant nepenthesin polypeptide comprises the sequence corresponding to the prepropolypeptide, propolypeptide, mature polypeptide, or polypeptide of SEQ ID NO: 1, 2, 3, 4, 5, or 6. In some embodiments, the recombinant nepenthesin polypeptide comprises the propolypeptide (e.g., proenzyme) of the sequence of SEQ ID NO: 1, 2, 3, 4, 5, or 6. In some embodiments, the recombinant nepenthesin polypeptide comprises the mature polypeptide of the sequence of SEQ ID NO: 1, 2, 3, 4, 5, or 6. In some embodiments, the recombinant nepenthesin polypeptide comprises an acid-activated proteolytically active polypeptide derived from or a subsequence of the 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 corresponding to the propolypeptide of 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 comprising the prepropolypeptide of SEQ ID NO: 1. In some embodiments, the recombinant neprosin polypeptide comprises an amino acid sequence comprising the propolypeptide of SEQ ID NO: 1. In some embodiments, the recombinant neprosin polypeptide comprises an amino acid sequence comprising the mature polypeptide of SEQ ID NO: 1. In some embodiments, the recombinant neprosin polypeptide comprises a polypeptide that is acid-activated and proteolytically active and that is derived from or comprises a subsequence of the sequence corresponding to SEQ ID NO: 1. In some embodiments, the mature neprosin polypeptide of SEQ ID NO: 1 has a molecular weight of from about 25.5 kDa to about 29 kDa, as determined particularly 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 corresponding to the propolypeptide of 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 comprising the prepropolypeptide of SEQ ID NO: 2. In some embodiments, the recombinant neprosin polypeptide comprises an amino acid sequence comprising the propolypeptide of SEQ ID NO: 2. In some embodiments, the recombinant neprosin polypeptide comprises an amino acid sequence comprising the mature polypeptide of SEQ ID NO: 2. In some embodiments, the recombinant neprosin polypeptide comprises an acid-activated proteolytically active polypeptide derived from or comprising a subsequence of the 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 corresponding to the propolypeptide of 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 comprising the prepropolypeptide of SEQ ID NO: 3. In some embodiments, the recombinant neprosin polypeptide comprises an amino acid sequence comprising the propolypeptide of SEQ ID NO: 3. In some embodiments, the recombinant neprosin polypeptide comprises an amino acid sequence comprising the mature polypeptide of SEQ ID NO: 3. In some embodiments, the recombinant neprosin polypeptide comprises an acid-activated proteolytically active polypeptide derived from or comprising a subsequence of the 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 corresponding to the propolypeptide of 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 comprising the prepropolypeptide of SEQ ID NO: 4. In some embodiments, the recombinant neprosin polypeptide comprises an amino acid sequence comprising the propolypeptide of SEQ ID NO: 4. In some embodiments, the recombinant neprosin polypeptide comprises an amino acid sequence comprising the mature polypeptide of SEQ ID NO: 4. In some embodiments, the recombinant neprosin polypeptide comprises an acid-activated proteolytic activity polypeptide derived from or comprising a subsequence of the 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 corresponding to the propolypeptide of 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 comprising the prepropolypeptide of SEQ ID NO: 5. In some embodiments, the recombinant neprosin polypeptide comprises an amino acid sequence comprising the propolypeptide of SEQ ID NO: 5. In some embodiments, the recombinant neprosin polypeptide comprises an amino acid sequence comprising the mature polypeptide of SEQ ID NO: 5. In some embodiments, the recombinant neprosin polypeptide comprises an acid-activated proteolytically active polypeptide derived from or comprising a subsequence of the 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 corresponding to the propolypeptide of 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 comprising the prepropolypeptide of SEQ ID NO: 6. In some embodiments, the recombinant neprosin polypeptide comprises an amino acid sequence comprising the propolypeptide of SEQ ID NO: 6. In some embodiments, the recombinant neprosin polypeptide comprises an amino acid sequence comprising the mature polypeptide of SEQ ID NO: 6. In some embodiments, the recombinant neprosin polypeptide comprises an acid-activated proteolytic activity polypeptide derived from or comprising a subsequence of the sequence corresponding to SEQ ID NO: 6.

[0204] Recombinant polynucleotides, expression vectors and host cells In a further aspect, the present disclosure provides an expression vector for the expression of a recombinant neprosin polypeptide in a suitable host cell, together with a recombinant polynucleotide encoding the recombinant neprosin polypeptide described herein.

[0205] As will be apparent to those skilled in the art, knowledge of the availability of protein sequences and the codons corresponding to various amino acids provides a description of any polynucleotide capable of encoding the polypeptide of interest. The degeneracy of the genetic code, where the same amino acid is encoded by alternative or synonymous codons, allows for the creation of a vast number of nucleic acids, all of which encode the recombinant neprosin polypeptide of the present disclosure. Thus, the present disclosure provides methods and compositions for generating any and all possible variant forms of a recombinant neprosin polynucleotide capable of encoding the recombinant neprosin polypeptide described herein by selecting combinations based on possible codon choices, and all such variant forms should be considered specifically disclosed for any of the polypeptides described herein that include the amino acid sequence of the recombinant neprosin polynucleotide described herein.

[0206] In some embodiments, the codons are preferably optimized for use by a selected host cell for protein production. In some embodiments, the polynucleotide encoding the recombinant neprosin polypeptide preferably uses codons that are used in mammalian cells, insect cells or fungal cells. In some embodiments, the codon-optimized polynucleotide encoding the recombinant neprosin polypeptide described herein contains 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 the prepropolypeptide, propolypeptide, mature polypeptide, or reference sequence corresponding to the polypeptide of SEQ ID NO: 1, wherein the amino acid sequence comprises substitutions with amino acids other than asparagine (N) at least at amino acid positions 68, 145, 152, or 253 or combinations thereof and their equivalent positions, where the amino acid positions are 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 or combinations 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 68, 145, 152, or 253 is A, respectively.

[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 the 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 68, or its equivalent position, where the amino acid position is compared 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 or its combination 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 the 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 its equivalent position, where the amino acid position is compared 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 or its combination 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 the reference sequence corresponding to the 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 amino acid position 152, or its equivalent position, where 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 or a combination thereof 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 the reference sequence corresponding to the 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 its equivalent position, where 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 or a combination thereof 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 the reference sequence corresponding to the prepropolypeptide, propolypeptide, mature polypeptide, or polypeptide of SEQ ID NO: 1, wherein the amino acid sequence comprises substitutions with amino acids other than asparagine (N) at least at amino acid positions 68 and 145, or their equivalent positions, where 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 position 68 and 145 or a combination thereof 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 the reference sequence corresponding to the prepropolypeptide, propolypeptide, mature polypeptide, or polypeptide of SEQ ID NO: 1, wherein the amino acid sequence comprises substitutions with amino acids other than asparagine (N) at least at amino acid positions 68 and 152, or their equivalent positions, where 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 position 68 and 152 or a combination thereof 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 the prepropolypeptide, propolypeptide, mature polypeptide, or polypeptide of SEQ ID NO:1, wherein the amino acid sequence comprises substitutions by amino acids other than asparagine (N) at least at amino acid positions 68 and 253, or their equivalent positions, where 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 position 68 and 253 or a combination thereof 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 the reference sequence corresponding to the prepropolypeptide, propolypeptide, mature polypeptide, or polypeptide of SEQ ID NO: 1, wherein the amino acid sequence comprises substitutions with amino acids other than asparagine (N) at least at amino acid positions 145 and 152, or their equivalent positions, where 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 or a combination thereof 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 the reference sequence corresponding to the prepropolypeptide, propolypeptide, mature polypeptide, or polypeptide of SEQ ID NO: 1, wherein the amino acid sequence comprises substitutions with amino acids other than asparagine (N) at least at positions 145 and 253, or their equivalent positions, where 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 or a combination thereof 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 the prepropolypeptide, propolypeptide, mature polypeptide, or polypeptide of SEQ ID NO:1, wherein the amino acid sequence comprises substitutions with amino acids other than asparagine (N) at least at amino acid positions 152 and 253, or their equivalent positions, where 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 position 152 and 253 or a combination thereof 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 the reference sequence corresponding to the prepropolypeptide, propolypeptide, mature polypeptide, or polypeptide of SEQ ID NO: 1, the amino acid sequence comprising substitutions by amino acids other than asparagine (N) at least at amino acid positions 68, 145, and 152, or their equivalent positions, 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 position 68, 145, and 152 or a combination thereof 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 the reference sequence corresponding to the prepropolypeptide, propolypeptide, mature polypeptide, or polypeptide of SEQ ID NO: 1, the amino acid sequence comprising substitutions by amino acids other than asparagine (N) at least at amino acid positions 68, 145, and 253, or their equivalent positions, 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 position 68, 145, and 253 or a combination thereof 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 the reference sequence corresponding to the prepropolypeptide, propolypeptide, mature polypeptide, or polypeptide of SEQ ID NO: 1, wherein the amino acid sequence comprises substitutions with amino acids other than asparagine (N) at least at amino acid positions 68, 152, and 253, or their equivalent positions, where 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 position 68, 152, and 253 or a combination thereof 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 the reference sequence corresponding to the prepropolypeptide, propolypeptide, mature polypeptide, or polypeptide of SEQ ID NO: 1, wherein the amino acid sequence comprises substitutions with amino acids other than asparagine (N) at least at amino acid positions 145, 152, and 253, or their equivalent positions, where 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 position 145, 152, and 253, or combinations thereof, 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 the reference sequence corresponding to the prepropolypeptide, propolypeptide, mature polypeptide, or polypeptide of SEQ ID NO: 1, wherein the amino acid sequence comprises substitutions with amino acids other than asparagine (N) at least at amino acid positions 68, 145, 152, and 253, or their equivalent positions, where 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 position 68, 145, 152, and 253 or a combination thereof 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 the reference sequence corresponding to the prepropolypeptide, propolypeptide, mature polypeptide, or polypeptide of SEQ ID NO: 1, wherein the amino acid sequence comprises at least substitutions N68A, N145A, N152A, or N253A, or their equivalent positions, and combinations thereof, where 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 the reference sequence corresponding to the prepropolypeptide, propolypeptide, mature polypeptide, or polypeptide of SEQ ID NO: 1, and the amino acid sequence comprises at least substitution N68A, or its equivalent position.

[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 the reference sequence corresponding to the prepropolypeptide, propolypeptide, mature polypeptide, or polypeptide of SEQ ID NO: 1, and the amino acid sequence comprises at least substitution N145A, or its equivalent position.

[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 the reference sequence corresponding to the prepropolypeptide, propolypeptide, mature polypeptide, or polypeptide of SEQ ID NO: 1, and the amino acid sequence comprises at least substitution N152A, or its equivalent position.

[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 the reference sequence corresponding to the prepropolypeptide, propolypeptide, mature polypeptide, or polypeptide of SEQ ID NO: 1, and the amino acid sequence comprises at least the substitution N253A, or their equivalent positions.

[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 the reference sequence corresponding to the prepropolypeptide, propolypeptide, mature polypeptide, or polypeptide of SEQ ID NO: 1, and the amino acid sequence comprises at least the substitutions N68A and N152A, or their equivalent positions.

[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 the reference sequence corresponding to the prepropolypeptide, propolypeptide, mature polypeptide, or polypeptide of SEQ ID NO: 1, and the amino acid sequence comprises at least the substitutions N68A and N253A, or their equivalent positions.

[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 the reference sequence corresponding to the prepropolypeptide, propolypeptide, mature polypeptide, or polypeptide of SEQ ID NO: 1, and the amino acid sequence comprises at least the substitutions N145A and N253A, or their equivalent positions.

[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 the reference sequence corresponding to the prepropolypeptide, propolypeptide, mature polypeptide, or polypeptide of SEQ ID NO: 1, and the amino acid sequence comprises at least the substitutions N152A and N253A, or their equivalent positions.

[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 the reference sequence corresponding to the prepropolypeptide, propolypeptide, mature polypeptide, or polypeptide of SEQ ID NO: 1, and the amino acid sequence comprises at least the substitutions N68A, N152A, and N253A, or their equivalent positions.

[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 the reference sequence corresponding to the prepropolypeptide, propolypeptide, mature polypeptide, or polypeptide of SEQ ID NO: 1, and the amino acid sequence comprises at least the substitutions N145A, N152A, and N253A, or their equivalent positions.

[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 the reference sequence corresponding to the prepropolypeptide, propolypeptide, mature polypeptide, or polypeptide of SEQ ID NO: 1, and the amino acid sequence comprises at least the substitutions N68A, N145A, N152A, and N253A, or their equivalent positions.

[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 the sequence corresponding to residues 381-26 of SEQ ID NO: 44, or its propolypeptide or mature polypeptide, and the amino acids at positions 68, 152, and / or 254 are other than 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 the sequence corresponding to residues 381-26 of SEQ ID NO: 46, or a polypeptide sequence comprising its pro-polypeptide or mature polypeptide, and the amino acid at position 68 is other than 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 the sequence corresponding to residues 381-26 of SEQ ID NO: 48, or a polypeptide sequence comprising its pro-polypeptide or mature polypeptide, and the amino acid at position 152 is other than 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 the sequence corresponding to residues 381-26 of SEQ ID NO: 50, or a polypeptide sequence comprising its pro-polypeptide or mature polypeptide, and the amino acid at position 254 is other than 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 the sequence corresponding to residues 381 to 26 of SEQ ID NO: 52, or a polypeptide sequence comprising its propolypeptide or mature polypeptide, wherein the amino acids at positions 68 and 254 are other than 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 amino acid residues 381 to 26 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 the propolypeptide of the polypeptide sequence comprising amino acid residues 381 to 26 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 the mature polypeptide of the polypeptide sequence comprising amino acid residues 381 to 26 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 356-105 of SEQ ID NO:8 or to a reference sequence corresponding to SEQ ID NO:8, the amino acid sequence comprising substitutions with amino acids other than asparagine (N) at least at amino acid positions 44, 121, 128, or 229 or combinations thereof, where 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 combinations 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 combinations thereof is A.

[0242] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant neprosin polypeptide comprising residues 356 to 105 of SEQ ID NO: 8, and the amino acid sequence comprises substitutions with amino acids other than asparagine (N) at least at amino acid positions 121, 128, or 229 or combinations thereof, wherein the amino acid positions are relative to SEQ ID NO: 8. In some embodiments, the encoded recombinant neprosin polypeptide comprises the amino acid sequence of SEQ ID NO: 8, and the amino acid sequence comprises substitutions with amino acids other than asparagine (N) at least at amino acid positions 44, 121, 128, or 229 or combinations 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 combinations 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 combinations 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 the reference sequence corresponding to SEQ ID NO: 8, and the amino acid sequence comprises substitutions with amino acids other than asparagine (N) at least at 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 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 or combinations thereof is A.

[0244] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant neprosin polypeptide comprising an acid-activated proteolytic activity 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 356-105 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 356-105 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, where 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 or a combination thereof is A.

[0246] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant neprosin polypeptide comprising an acid-activated proteolytic activity 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 356-105 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 nepocin 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 356-105 of SEQ ID NO: 8 or to a reference sequence corresponding to SEQ ID NO: 8, the amino acid sequence comprising a substitution by an amino acid other than asparagine (N) at least at amino acid position 128, where 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 or a combination thereof is A.

[0248] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant nepocin polypeptide comprising an acid-activated proteolytic activity polypeptide derived from or comprising a subsequence of SEQ ID NO: 14. In some embodiments, the encoded recombinant nepocin polypeptide comprises an amino acid sequence comprising residues 356-105 of SEQ ID NO: 14. In some embodiments, the encoded recombinant nepocin 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 356-105 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, where 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 or a combination thereof is A.

[0250] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant neprosin polypeptide comprising an acid-activated proteolytic activity 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 356-105 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 nepocin 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 356-105 of SEQ ID NO: 8 or to a reference sequence corresponding to SEQ ID NO: 8, the amino acid sequence comprising substitutions with amino acids other than asparagine (N) at least at 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 nepocin polypeptide comprising an acid-activated proteolytic activity polypeptide derived from or comprising a subsequence of SEQ ID NO: 18. In some embodiments, the encoded recombinant nepocin polypeptide comprises an amino acid sequence comprising residues 356-105 of SEQ ID NO: 18. In some embodiments, the encoded recombinant nepocin 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 356 to 105 of SEQ ID NO: 8, or to a reference sequence corresponding to SEQ ID NO: 8, wherein the amino acid sequence comprises substitutions with amino acids other than asparagine (N) at least at amino acid positions 44, 128, and 229, where 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 proteolytic activity 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 356 to 105 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 nepocin 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 356 to 105 of SEQ ID NO: 8, or to a reference sequence corresponding to SEQ ID NO: 8, wherein the amino acid sequence comprises substitutions with amino acids other than asparagine (N) at least at amino acid positions 44, 121, 128, and 229, where 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 nepocin polypeptide comprising an acid-activated proteolytic activity polypeptide derived from or comprising a subsequence of SEQ ID NO: 22. In some embodiments, the encoded recombinant nepocin polypeptide comprises an amino acid sequence comprising residues 356 to 105 of SEQ ID NO: 22. In some embodiments, the encoded recombinant nepocin 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 nepocin 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 356 to 105 of SEQ ID NO: 8, or to a reference sequence corresponding to SEQ ID NO: 8, wherein the amino acid sequence comprises substitutions with amino acids other than asparagine (N) at least at amino acid positions 44 and 229, where 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 nepocin polypeptide comprising an acid-activated proteolytic activity polypeptide derived from SEQ ID NO: 32 or a subsequence thereof. In some embodiments, the encoded recombinant nepocin polypeptide comprises an amino acid sequence comprising residues 356 to 105 of SEQ ID NO: 32. In some embodiments, the encoded recombinant nepocin 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 nepocin 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 residues 356 to 105 of SEQ ID NO: 8, or to a sequence corresponding to SEQ ID NO: 8.

[0260] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant nepocin polypeptide comprising an acid-activated proteolytic activity polypeptide derived from or comprising a subsequence of SEQ ID NO: 8. In some embodiments, the encoded recombinant nepocin polypeptide comprises an amino acid sequence comprising residues 356-105 of SEQ ID NO: 8. In some embodiments, the encoded recombinant nepocin 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 nepocin polypeptide comprising an amino acid sequence comprising residues 451-96 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 nepocin polypeptide comprising a pro-polypeptide of an amino acid sequence comprising residues 451-96 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 nepocin polypeptide comprising a mature polypeptide of an amino acid sequence comprising residues 451-96 of SEQ ID NO: 58, 60, 62, 64, 66, 68, or 70.

[0262] In some embodiments, the recombinant polynucleotide comprises a reference polynucleotide sequence corresponding to nucleotide residues 1068-313 of SEQ ID NO: 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, or 33, or 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 the polynucleotide sequence corresponding to SEQ ID NO: 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, or 33, and the recombinant polynucleotide encodes a nepocin polypeptide.

[0263] In some embodiments, the recombinant polynucleotide comprises nucleotide residues 1068-313 of 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 a signal peptide, as described herein. In some embodiments, the encoded signal sequence or signal peptide is functional in the host cell in which it is or will be used for the expression of the neprosin polypeptide. In some embodiments, the encoded signal sequence or signal peptide is fused to the pro-polypeptide form of recombinant neprosin to form a pre-pro-polypeptide. In some embodiments, the encoded signal sequence or signal peptide is fused to a polypeptide comprising the mature active form of recombinant neprosin. In some embodiments, the encoded signal sequence can 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 comprising a fusion protein. In some embodiments, the encoded recombinant neprosin polypeptide can be fused to various polypeptide sequences described above. In some embodiments, the fusion protein of the recombinant neprosin polypeptide comprises a glycine-histidine or 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 encoding a recombinant neprosin polypeptide comprising SEQ ID NO: 55, 57, 59, 61, 63, 65, 67, or 69.

[0268] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant neprosin polypeptide comprising a SEQ ID NO.

[0269] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant neprosin polypeptide having an amino acid sequence with 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, 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 corresponding to the propolypeptide of 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 nepocin polypeptide comprising an amino acid sequence comprising the prepropolypeptide of SEQ ID NO: 1. In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant nepocin polypeptide comprising an amino acid sequence comprising the propolypeptide of SEQ ID NO: 1. In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant nepocin polypeptide comprising an amino acid sequence comprising the mature polypeptide of SEQ ID NO: 1. In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant nepocin polypeptide comprising an acid-activated proteolytic activity polypeptide derived from or comprising a subsequence of the sequence corresponding to SEQ ID NO: 1.

[0274] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant nepocin 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 nepocin 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 propolypeptide of 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 comprising the prepropolypeptide of SEQ ID NO: 2. In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant neprosin polypeptide comprising an amino acid sequence comprising the propolypeptide of SEQ ID NO: 2. In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant neprosin polypeptide comprising an amino acid sequence comprising the mature polypeptide of SEQ ID NO: 2. In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant neprosin polypeptide comprising an acid-activated proteolytic activity polypeptide derived from or comprising a subsequence of the 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 corresponding to the propolypeptide of 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 comprising the prepropolypeptide of SEQ ID NO: 3. In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant neprosin polypeptide comprising an amino acid sequence comprising the propolypeptide of SEQ ID NO: 3. In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant neprosin polypeptide comprising an amino acid sequence comprising the mature polypeptide of SEQ ID NO: 3. In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant neprosin polypeptide comprising an acid-activated proteolytic activity polypeptide derived from or comprising a subsequence of the 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 corresponding to the propolypeptide of 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 comprising the prepropolypeptide of SEQ ID NO: 4. In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant neprosin polypeptide comprising an amino acid sequence comprising the propolypeptide of SEQ ID NO: 4. In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant neprosin polypeptide comprising an amino acid sequence comprising the mature polypeptide of SEQ ID NO: 4. In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant neprosin polypeptide comprising an acid-activated proteolytic activity polypeptide derived from or comprising a subsequence of the 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 corresponding to the propolypeptide of 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 comprising the prepropolypeptide of SEQ ID NO: 5. In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant neprosin polypeptide comprising an amino acid sequence comprising the propolypeptide of SEQ ID NO: 5. In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant neprosin polypeptide comprising an amino acid sequence comprising the mature polypeptide of SEQ ID NO: 5. In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant neprosin polypeptide comprising an acid-activated proteolytic activity polypeptide derived from or comprising a subsequence of the 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 corresponding to the propolypeptide of 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 comprising the prepropolypeptide of SEQ ID NO: 6. In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant neprosin polypeptide comprising an amino acid sequence comprising the propolypeptide of SEQ ID NO: 6. In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant neprosin polypeptide comprising an amino acid sequence comprising the mature polypeptide of SEQ ID NO: 6. In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant neprosin polypeptide comprising an acid-activated proteolytic activity polypeptide derived from or comprising a subsequence of the sequence corresponding to SEQ ID NO: 6.

[0294] In another aspect, the present disclosure further provides an expression vector comprising a recombinant polynucleotide encoding a recombinant nepocin polypeptide described herein, for example, for the expression of the encoded recombinant nepocin polypeptide. In some embodiments, the expression vector comprises one or more control sequences for regulating the expression of the nepocin polynucleotide and / or polypeptide. In some embodiments, the control sequences include, inter alia, a promoter, a leader sequence, a polyadenylation sequence, a propeptide sequence, a signal peptide sequence, and a transcription terminator. In particular, control sequences such as a promoter, a leader sequence, a polyadenylation sequence, a propeptide sequence, a signal peptide sequence, and a transcription terminator are control sequences effective in a selected host cell, such as a mammalian cell, an insect cell, or a fungal cell.

[0295] In some embodiments, promoters suitable for directing transcription of nucleic acid constructs in mammalian cells include, among others, the SV40 promoter, or promoters derived from cytomegalovirus (CMV), chicken beta-actin (CAG), or elongation factor (EF)-1. In some embodiments, promoters suitable for directing transcription of nucleic acid constructs in insect cells include, among others, baculovirus promoters (e.g., P10 and polyhedron promoters), the OpIE2 promoter, and the Nephotettix cincticeps actin promoter. In some embodiments, promoters suitable for directing transcription in fungal cells include, among others, promoters of the AOX1 gene, TEF1 gene, POX2 gene, TDH3 gene, PGK gene, ADH1 gene, together with 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).Other fungal promoters include those obtained from the genes of 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, for example, WO96 / 00787), as well as the NA2 - tpi promoter (a hybrid of the promoters from the genes of Aspergillus niger neutral alpha - amylase and Aspergillus oryzae triose phosphate isomerase), and mutants, truncates, and hybrids thereof, but are not limited thereto. Exemplary yeast cell promoters can be derived from the genes of 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 finds use for the expression of the neprosin polypeptide. Any suitable terminator that is functional in the selected host cell finds use in the expression of the neprosin polypeptide. By way of non-limiting example, transcription terminators include those derived from the human β-globin gene, SV40, hGH, IE1 terminator, and BGH. Exemplary fungal transcription terminators include, among others, those from the genes of Aspergillus oryzae TAKA amylase, Aspergillus niger glucoamylase, Aspergillus nidulans anthranilate synthase, Aspergillus niger alpha-glucosidase, and Fusarium oxysporum trypsin-like protease. Exemplary terminators for yeast host cells can be obtained from the genes of 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., the untranslated region of the mRNA important for translation by the host cell). In some embodiments, the leader sequence is operably linked to the 5' end of the nucleic acid sequence encoding the neprosin polypeptide. Any suitable leader sequence that is functional in the selected host cell finds use in the expression of the recombinant neprosin polypeptide. Exemplary leader sequences for mammalian and insect cells include, inter alia, leader sequences of genes being expressed (e.g., heat shock proteins, myosin, BIP immunoglobulin binding protein, GRP glucose-regulated protein, etc.), viral leader sequences (e.g., EMC virus), and synthetic leader sequences such as hTEE-658 and those described in Cao et al., Nature Commun., 2021, 12:4138, which is incorporated herein by reference. Exemplary leaders for fungal expression include, inter alia, those from the genes of Aspergillus oryzae TAKA amylase and Aspergillus nidulans triose phosphate isomerase. Suitable leaders for yeast host cells are obtained from the genes of 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 array is also a polyadenylation array (i.e., an array that is operably linked to the 3' end of a nucleic acid sequence and, when transcribed, is recognized by the host cell as a signal for adding polyadenosine residues to the transcribed mRNA). Any suitable polyadenylation array that is functional in the selected host cell can be found for use in the present invention. Exemplary polyadenylation arrays for mammalian and insect cells include, among others, the polyadenylation arrays of human and mouse alpha-globin, mouse kappa light chain, chicken ovalbumin, the SV40 gene, as well as synthetic polyA arrays (see, e.g., Clerici et al., eLife, 2017, 6:e33111). Exemplary polyadenylation arrays for fungal host cells include, but are not limited to, the genes of Aspergillus oryzae TAKA amylase, Aspergillus niger glucoamylase, Aspergillus nidulans anthranilate synthase, Fusarium oxysporum trypsin-like protease, and Aspergillus niger alpha-glucosidase. Useful polyadenylation arrays for yeast host cells are known in the art (see, e.g., Guo and Sherman, Mol. Cell. Bio., 1995, 15:5983-5990).

[0299] In some embodiments, as described herein, the control sequence is also a signal peptide (i.e., a coding region that encodes an amino acid sequence linked to the amino terminus of a polypeptide and directs the encoded polypeptide into the secretory pathway of the cell). In some embodiments, the 5' end of the coding sequence of the nucleic acid sequence uniquely contains a signal peptide coding region that is naturally linked in-frame with a segment of the coding region encoding the secreted polypeptide. Alternatively, in some embodiments, the 5' end of the coding sequence contains a signal peptide coding region that is exogenous to the coding sequence, i.e., a heterologous signal sequence. Any suitable signal peptide coding region that directs the expressed polypeptide into the secretory pathway of the selected host cell is contemplated for use in the expression of the engineered polypeptide(s). 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 disclosure provides an expression vector comprising a recombinant polynucleotide encoding a recombinant neprosin polypeptide and, depending on the type of host into which it is to be introduced, one or more expression control regions such as a promoter and a terminator, an origin of replication, etc. In some embodiments, the various nucleic acid and control sequences described herein are joined together (i.e., operably linked) to produce a recombinant expression vector that contains one or more convenient restriction sites to allow for the insertion or substitution of a nucleic acid sequence encoding a recombinant neprosin polypeptide at such sites.

[0301] A recombinant expression vector can be any suitable vector (e.g., plasmid or virus) that can be readily subjected to recombinant DNA procedures and can result in the expression of a polynucleotide sequence encoding neprosin. The choice of vector typically depends on the compatibility of the vector with the host cell into which it is to be introduced. The vector can be a linear or 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 does not depend on chromosomal replication, such as a plasmid, episomal element, minichromosome or artificial chromosome). The vector can contain any means for ensuring self - replication. In some alternative embodiments, the vector is a vector that integrates into the genome when introduced into the host cell and is replicated along with the chromosome(s) into which it has integrated. Further, in some embodiments, a single vector or plasmid, or two or more vectors or plasmids and / or transposons that together contain the total DNA to be introduced into the genome of the host cell are utilized.

[0303] In some embodiments, the expression vector contains one or more selectable markers that enable easy selection of the transformed cells. A "selectable marker" is a gene whose product provides, for example, biocide or virus resistance, resistance to heavy metals, prototrophy to auxotrophic strains, etc. Suitable markers for mammalian cells include, depending on the cell type, among others, 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). Suitable markers for insect cells include, depending on the cell type, among others, puromycin acetyltransferase, aminoglycoside phosphotransferase, and hygromycin-B-phosphotransferase (hygromycin resistance). 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., derived from A. nidulans or A. orzyae), argB (ornithine carbamoyltransferase), bar (phosphinothricin acetyltransferase; e.g., derived from S. hygroscopicus), hph (hygromycin phosphotransferase), niaD (nitrate reductase), pyrG (orotidine-5'-phosphate decarboxylase; e.g., derived from A. nidulans or A. orzyae), sC (sulfate adenylyltransferase), and trpC (anthranilate synthase), and equivalents thereof.

[0304] In a further aspect, the present disclosure provides a host cell comprising the 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 the recombinant neprosine polypeptide described herein. In some embodiments, the recombinant neprosine polypeptide expressed in the host cell is in the prepropolypeptide or preproenzyme form of the neprosine polypeptide. In some embodiments, the recombinant neprosine polypeptide expressed in the host cell is in the propolypeptide or proenzyme form of the neprosine polypeptide. In some embodiments, the recombinant neprosine polypeptide expressed in the host cell is the mature neprosine polypeptide.

[0306] In some embodiments, the host cell comprises a polynucleotide sequence encoding a recombinant neprosine polypeptide, and the encoded recombinant neprosine polypeptide comprises a signal sequence. In some embodiments, the signal sequence is selected to be appropriate for the host cell. 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 neprosine polypeptide. In some embodiments, the host cell expresses the recombinant neprosine polypeptide in a soluble form.

[0307] In some embodiments, the host cell expresses a glycosylated recombinant nephrosin polypeptide. In some embodiments, the host cell expresses a recombinant nephrosin polypeptide that is glycosylated but lacks glycosylation at at least one of the glycosylation sites described herein. In some embodiments, the host cell expresses a recombinant nephrosin polypeptide that is non-glycosylated. In some embodiments, the glycosylation pattern of the recombinant nephrosin 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, among others, 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. In some embodiments, the mammalian host cell is deficient in glycosylation (see, e.g., Esko et al., Glycosylation Mutants of Cultured Mammalian Cells. 2017. In: Varki A, Cummings RD, Esko JD, et al., editors. 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 nephrosin 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 lepidopteran or dipteran insect cell. Exemplary insect host cells include, among others, Sf9 cells, Sf21 cells, Schneider 2 cells, and BTI-TN-5B1-4 (High Five) cells. In some embodiments, the insect host cell contains a humanized glycosylation pathway enzyme (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 codon-optimized for expression 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 yeast. 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 T. Trichoderma or Myceliophthora fungal cell. Exemplary fungal host cells include, among others, Pichia pastoris, Yarrowia lipolytica, Kluyveromyces marxianus, Kluyveromyces lactis, Aspergillus niger, Aspergillus oryzae, Aspergillus fumigatus Trichoderma reesei. Neurospora crassa, Mucor circinelloides, Penicillium chrysogenum T. reesei, Trichoderma harzianum, Saccharomyces cerevisiae or Myceliophthora thermophile. 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 for the expression of the recombinant neprosin polypeptide is Pichia pastoris or Saccharomyces cerevisiae.

[0311] In some embodiments, any suitable method for introducing the polynucleotide for the expression of the recombinant neprosin polypeptide into the cell is contemplated for use for the purposes of this specification. Suitable techniques include, but are not limited to, electroporation, particle bombardment, liposome-mediated transfection, calcium chloride transfection and protoplast fusion.

[0312] Expression and processing of the recombinant neprosin polypeptide In another aspect, the present disclosure provides a method for expressing the recombinant nepocin polypeptide described herein. In some embodiments, the present disclosure further provides the processing of the expressed recombinant nepocin polypeptide for preparing a proteolytically active recombinant nepocin polypeptide, particularly the proteolytically active form of the mature nepocin polypeptide.

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

[0315] As described herein, in some embodiments, the mammalian cell is a rodent cell or a human cell. Exemplary mammalian cells for use as host cells include Expi293, HeLa, U2OS, A549, HT1080, CAD, P19, NIH 3T3, 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 deficient in glycosylation (see, e.g., Esko et al., Glycosylation Mutants of Cultured Mammalian Cells. 2017. In: Varki A, Cummings RD, Esko JD, et al., editors. Essentials of Glycobiology, 3rd Ed., Chapter 49, Cold Spring Harbor (NY): Cold Spring Harbor Laboratory Press; 2015-2017).

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

[0317] As described above, in some embodiments, the insect cell is a lepidopteran or dipteran insect cell. Exemplary insect cells for use 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 a humanized glycosylation pathway enzyme (see, e.g., Yee et al., Ind. Eng. Chem. Res., 2018, 57:10061-10070).

[0318] In some embodiments, a method of producing a recombinant nepocin polypeptide comprises culturing a fungal cell comprising an expression vector comprising a recombinant polynucleotide encoding a recombinant nepocin polypeptide as described herein under suitable culture conditions such that the encoded recombinant nepocin 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, Hansemula, 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 cells for use as host cells are Pichia pastoris, Yarrowia lipolytica, Kluyveromyces marxianus, Kluyveromyces lactis, Aspergillus niger, Aspergillus oryzae, Aspergillus fumigatus Trichoderma reesei, Neurospora crassa, Mucor circinelloides, Penicillium chrysogenum T. reesei, Trichoderma harzianum, Saccharomyces cerevisiae and Myceliophthora thermophile.

[0320] Suitable culture media and growth conditions for mammalian, insect, and fungal cells for expressing heterologous proteins are well known in the art. Exemplary culture media for mammalian cells include, inter alia, Eagle's Minimum Essential Medium (EMEM), Dulbecco's Modified Eagle Medium (DMEM), Iscove's Modified Dulbecco Medium (IMDM), Hybri-Care Medium, McCoy's 5A, and RPMI-1640, Ham's nutrient mixtures, and DMEM / F12 medium. Such media formulations are described, inter alia, in ATCC Animal Cell Culture Guide, 2022; Phelan, K. and May, K.M., 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 Ed., Wiley-Blackwell (2016).

[0321] Exemplary culture media for insect cells include, among others, 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 Drosophila medium. Such media formulations are described, among others, in Agathos, S.N. “Development of serum-free media for lepidopteran insect cell lines,” In Baculovirus Expression Protocols, 2nd Ed., D. W. Murhammer, ed., p. 155-185, Humana Press, Totowa, NJ (2007); and KaBer et al., Electronic Journal of Biotechnology, 2022, 56:54-64.

[0322] The culture medium for fungal cells can vary depending on the type of fungal cells used for the expression of the recombinant neprosin polypeptide. Exemplary media for Pichia pastoris include Rich Defined Medium (RDM) or Buffered Complex Glycerol Medium (BMGY) (see Mathews et al., Biotechnol Bioeng, 2018, 115(1):103-113); YNB / YND medium (Yeast Nitrogen Base), YPM medium or BMM (Buffered Minimal Medium) (see, for example, Lin-Cereghino et al., “Expression of protein in Pichia pastoris,” In Expression systems: methods express., Dyson M, Durocher Y (ed), p 123-145, Scion Publishing, Banbury, United Kingdom (2007)). Exemplary culture media for S. cerevisiae include, inter alia, YPD and YEPD media (see, for example, Dymond, JS., Methods Enzymol., 2013, 533:191-204).

[0323] In some embodiments, the method further comprises isolating the expressed recombinant neprosin polypeptide. In some embodiments, the method further comprises purifying the expressed recombinant neprosin polypeptide. In some embodiments, the isolated or purified neprosin polypeptide comprises the prepropolypeptide (e.g., preproenzyme) form of the recombinant neprosin polypeptide. In some embodiments, the isolated or purified neprosin polypeptide comprises the propolypeptide (e.g., proenzyme) form of the recombinant neprosin polypeptide. In some embodiments, the isolated or purified neprosin polypeptide comprises the mature form of the recombinant neprosin polypeptide.

[0324] In some embodiments, the recombinant neprosin polypeptide expressed and produced in a host cell is isolated and / or purified from the cells and / or the culture medium using any one or more of well-known techniques for protein purification, including, among other things, detergent treatment, sonication, filtration, salting out, ultracentrifugation, and chromatography. Chromatographic techniques for the isolation and purification of the neprosin polypeptide include, among other things, reverse phase chromatography, high performance liquid chromatography, ion exchange chromatography, hydrophobic interaction chromatography, size exclusion chromatography, gel electrophoresis, and affinity chromatography. The conditions for purifying a particular enzyme depend, in part, on factors such as net charge, hydrophobicity, hydrophilicity, molecular weight, molecular shape, etc., and will be apparent to those skilled in the art. In some embodiments, affinity techniques can be used to isolate the recombinant neprosin polypeptide. For affinity chromatography purification, an antibody that specifically binds to the neprosin polypeptide of interest can be found for use. In some embodiments, the recombinant neprosin polypeptide is isolated and / or purified using a polypeptide tag present on the protein, such as a His-tag.

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

[0327] In some embodiments, the method is used to express a glycosylated recombinant nepocin polypeptide. In some embodiments, the method is used to express a prepropolypeptide of a glycosylated recombinant nepocin polypeptide. In some embodiments, the method is used to express a propolypeptide of a glycosylated recombinant nepocin polypeptide. In some embodiments, the method is used to express a mature polypeptide of a glycosylated recombinant nepocin polypeptide. In some embodiments, the expressed recombinant nepocin polypeptide is glycosylated at one or more positions 68, 145, 152 or 253 or combinations thereof, and the amino acid positions are relative to SEQ ID NO: 1. In some embodiments, the expressed recombinant nepocin polypeptide is glycosylated at one or more positions 44, 121, 128 or 229 or combinations thereof, and the amino acid positions are relative to SEQ ID NO: 8.

[0328] In some embodiments, targeting glycosylation can involve using a recombinant polynucleotide encoding a recombinant nepocin polypeptide having a mutation at amino acid position 68, 145, 152 or 253 or combinations thereof, relative to SEQ ID NO: 1, to express a recombinant nepocin polypeptide glycosylated at defined positions. In some embodiments, the expressed recombinant nepocin polypeptide is glycosylated at least at amino acid position 68. In some embodiments, the expressed recombinant nepocin polypeptide is glycosylated at least at amino acid position 145. In some embodiments, the expressed recombinant nepocin polypeptide is glycosylated at least at amino acid position 152. In some embodiments, the expressed recombinant nepocin polypeptide is glycosylated at least at amino acid position 253.

[0329] In some embodiments, the expressed recombinant nepocin polypeptide is non-glycosylated. In some embodiments, the expressed recombinant nepocin is a non-glycosylated prepropolypeptide. In some embodiments, the expressed recombinant nepocin is a non-glycosylated propolypeptide. In some embodiments, the expressed recombinant nepocin is a non-glycosylated mature polypeptide.

[0330] As described herein, the method is used to express a recombinant nepocin polypeptide comprising a prepropolypeptide, propolypeptide or mature form of the recombinant nepocin polypeptide in mammalian cells, insect cells or fungal cells. In some preferred embodiments, the method is used to express a recombinant nepocin polypeptide in fungal cells, particularly Pichia pastoris.

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

[0332] In some embodiments, the expressed recombinant neprosin polypeptide lacks glycosylation at one or more or a combination thereof of positions 44, 121, 128, or 229, where the amino acid positions are relative to SEQ ID NO: 8. In some embodiments, the expressed recombinant neprosin polypeptide lacks glycosylation at least at position 44 or its equivalent position. In some embodiments, the expressed recombinant neprosin polypeptide lacks glycosylation at least at position 121, or its equivalent position. In some embodiments, the expressed recombinant neprosin polypeptide lacks glycosylation at least at position 128, or its equivalent position. In some embodiments, the expressed recombinant neprosin polypeptide lacks glycosylation at least at position 229, or its equivalent position. In some embodiments, the expressed recombinant neprosin polypeptide lacks glycosylation at least at positions 44 and 128, or their equivalent positions. In some embodiments, the expressed recombinant neprosin polypeptide lacks glycosylation at least at positions 44 and 229, or their equivalent positions. In some embodiments, the expressed recombinant neprosin polypeptide lacks glycosylation at least at positions 128 and 229, or their equivalent positions. In some embodiments, the expressed recombinant neprosin polypeptide lacks glycosylation at least at positions 12 and 229, or their equivalent positions.

[0333] In some embodiments, the recombinant neprosin polypeptide expressed has 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 356 - 105 of SEQ ID NO: 8 or to a reference sequence corresponding to SEQ ID NO: 8, and the amino acid sequence includes substitutions with amino acids other than asparagine (N) at at least amino acid positions 44, 121, 128, or 229 or combinations thereof, where 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 combinations 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 combinations thereof is A.

[0334] In some embodiments, the expressed recombinant neprosin polypeptide comprises an amino acid sequence comprising residues 356-105 of SEQ ID NO: 8, and the amino acid sequence comprises substitutions with amino acids other than asparagine (N) at at least amino acid positions 121, 128, or 229 or combinations thereof, where 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, and the amino acid sequence comprises substitutions with amino acids other than asparagine (N) at at least amino acid positions 44, 121, 128, or 229 or combinations thereof, where 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 combinations 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 combinations 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 356-105 of SEQ ID NO: 8 or to a reference sequence corresponding to SEQ ID NO: 8, and the amino acid sequence comprises a substitution with an amino acid other than asparagine (N) at at least amino acid position 44, where 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 or combinations thereof is A.

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

[0337] In some embodiments, the expressed recombinant nepocin 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 356-105 of SEQ ID NO: 8 or to a reference sequence corresponding to SEQ ID NO: 8, the amino acid sequence comprising a substitution by an amino acid other than asparagine (N) at least at amino acid position 121, where 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 or a combination thereof is A.

[0338] In some embodiments, the expressed recombinant neprosin polypeptide comprises an acid-activated proteolytic activity polypeptide derived from or comprising a subsequence of SEQ ID NO: 12. In some embodiments, the expressed recombinant neprosin polypeptide comprises an amino acid sequence comprising residues 356-105 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 an amino acid sequence comprising residues 356-105 of SEQ ID NO: 12 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 356-105 of SEQ ID NO: 8 or to a reference sequence corresponding to SEQ ID NO: 8, the amino acid sequence comprising a substitution by an amino acid other than asparagine (N) at least at amino acid position 128, where 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 or a combination thereof is A.

[0340] In some embodiments, the expressed recombinant neprosin polypeptide comprises an acid-activated proteolytic activity polypeptide derived from or comprising a subsequence of SEQ ID NO: 14. In some embodiments, the expressed recombinant neprosin polypeptide comprises an amino acid sequence comprising residues 356-105 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 an amino acid sequence comprising residues 356-105 of SEQ ID NO: 14 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 the sequence corresponding to residues 356-105 of SEQ ID NO: 8 or to the sequence corresponding to SEQ ID NO: 8, and the amino acid sequence comprises a substitution with an amino acid other than asparagine (N) at at least amino acid position 229 or its equivalent position, where 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 or a combination thereof is A.

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

[0343] In some embodiments, the recombinant neprosin polypeptide expressed has 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 356-105 of SEQ ID NO: 8, or to a reference sequence corresponding to SEQ ID NO: 8, and the amino acid sequence comprises substitutions by amino acids other than asparagine (N) at least at amino acid positions 121 and 229, where the amino acid positions are relative to SEQ ID NO: 8. In some embodiments, the amino acid substitutions or amino acid residues at each of positions 121 and 229 are 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 121 and 229, or a combination thereof, is A.

[0344] In some embodiments, the expressed recombinant neprosin polypeptide comprises an acid-activated proteolytic activity polypeptide derived from or comprising a subsequence of SEQ ID NO: 18. In some embodiments, the expressed recombinant neprosin polypeptide comprises an amino acid sequence comprising residues 356-105 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 amino acid sequence comprising residues 356-105 of SEQ ID NO: 18, or the recombinant neprosin polypeptide 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 356-105 of SEQ ID NO: 8, or to a reference sequence corresponding to SEQ ID NO: 8, wherein the amino acid sequence comprises substitutions with amino acids other than asparagine (N) at least at amino acid positions 44, 128, and 229, where 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 position 44, 128, and 229 or combinations thereof is A.

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

[0347] In some embodiments, the expressed recombinant nepocin 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 356 to 105 of SEQ ID NO: 8, or to a reference sequence corresponding to SEQ ID NO: 8, the amino acid sequence comprising substitutions by amino acids other than asparagine (N) at least at amino acid positions 44, 121, 128, and 229, where 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 position 44, 121, 128, and 229, or combinations thereof, is A.

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

[0349] In some embodiments, the expressed recombinant neprosine 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 356-105 of SEQ ID NO: 8 or to a reference sequence corresponding to SEQ ID NO: 8, the amino acid sequence comprising substitutions by amino acids other than asparagine (N) at least at amino acid positions 44 and 229, where 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 position 44 and 229 or a combination thereof is A.

[0350] In some embodiments, the expressed recombinant neprosin polypeptide comprises an acid-activated proteolytic activity polypeptide derived from SEQ ID NO: 32 or comprising a subsequence thereof. In some embodiments, the expressed recombinant neprosin polypeptide comprises an amino acid sequence comprising residues 356-105 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 an amino acid sequence comprising residues 356-105 of SEQ ID NO: 32 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 356-105 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 proteolytic activity polypeptide derived from SEQ ID NO: 8 or comprising a subsequence thereof. In some embodiments, the expressed recombinant neprosin polypeptide comprises an amino acid sequence comprising residues 356-105 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 an amino acid sequence comprising residues 356-105 of SEQ ID NO: 8 or comprising SEQ ID NO: 22 is glycosylated.

[0353] In some embodiments, the expressed recombinant nepocin polypeptide comprises an amino acid sequence comprising residues 381-26 of SEQ ID NO: 44, 46, 48, 50, or 52. In some embodiments, the expressed recombinant nepocin polypeptide comprises a propolypeptide contained in residues 381-26 of SEQ ID NO: 44, 46, 48, 50, or 52. In some embodiments, the expressed recombinant nepocin polypeptide comprises a mature nepocin polypeptide contained in residues 381-26 of SEQ ID NO: 44, 46, 48, 50, or 52. In some embodiments, the expressed recombinant nepocin 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 nepocin polypeptide comprises an amino acid sequence comprising residues 451-96 of SEQ ID NO: 58, 60, 62, 64, 66, 68, or 70. In some embodiments, the expressed recombinant nepocin polypeptide comprises a propolypeptide contained in residues 451-96 of SEQ ID NO: 58, 60, 62, 64, 66, 68, or 70. In some embodiments, the expressed recombinant nepocin polypeptide comprises a mature nepocin polypeptide contained in residues 451-96 of SEQ ID NO: 58, 60, 62, 64, 66, 68, or 70. In some embodiments, the expressed recombinant nepocin polypeptide comprises an amino acid sequence comprising SEQ ID NO: 54, 56, 58, 60, 62, 64, 66, 68, or 70.

[0355] In some embodiments, a method for preparing a mature neprosin polypeptide comprises processing a full-length neprosin polypeptide or a neprosin prepropolypeptide or a propolypeptide prepared from an expressed neprosin as described herein; or processing a full-length neprosin polypeptide or a neprosin prepropolypeptide or a propolypeptide of a recombinant neprosin variant as described herein to an acidic pH under conditions suitable for the formation of the mature neprosin polypeptide. In some embodiments, the processing is at a pH of about 2 to about 6. In some embodiments, the processing is at a pH of about 2 to about 4.5. In some embodiments, the processing is at a pH of about 2 to about 3. In some embodiments, the method for 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, as determined particularly by SDS-PAGE.

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

[0357] In some embodiments, the present disclosure provides a mature, proteolytically active recombinant neprosin polypeptide prepared by acid activation of a prepropolypeptide or a propolypeptide of a recombinant neprosin polypeptide as described herein.

[0358] Pharmaceuticals and dietary / nutritional compositions In a further aspect, the recombinant nepocin is formulated as a composition. In some embodiments, the composition comprises a recombinant prepropolypeptide or preproenzyme form of the nepocin polypeptide, a propolypeptide or proenzyme form of the nepocin polypeptide, or a mature nepocin polypeptide as described herein. In some embodiments, the recombinant nepocin polypeptide in the composition is prepared from an expressed recombinant nepocin polypeptide or recombinant nepocin polypeptide variant comprising a prepropolypeptide, propolypeptide or mature polypeptide form of a variant as described herein. In some embodiments, the mature nepocin polypeptide in the composition has a molecular weight of from about 25.5 kDa to about 29 kDa, as determined, for example, by SDS-PAGE. In some embodiments, the recombinant nepocin polypeptide in the composition is prepared from a recombinant nepocin polypeptide expressed or produced in mammalian cells, insect cells or fungal cells.

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

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

[0361] In some embodiments, the recombinant neprosin is formulated as a pharmaceutical or dietary composition. Depending on the mode of administration, the composition contains a therapeutically effective amount of the recombinant neprosin polypeptide and can be in solid, semi-solid or liquid form. The term "pharmaceutically acceptable" means approved by a regulatory agency of the Federal or State government or listed in the U.S. Pharmacopeia 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 with which the therapeutic agent is administered. Such pharmaceutical carriers can be sterile liquids such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Aqueous solutions of salts and aqueous dextrose and glycerol solutions can 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, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene glycol, water, ethanol and the like. The composition can also contain, if desired, minor amounts of wetting or emulsifying agents or pH buffers. Such compositions can take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained release formulations and other forms. Examples of suitable pharmaceutical carriers are described in "Remington’s Pharmaceutical Sciences" by E. W. Martin, which is hereby incorporated by reference in its entirety. Such compositions will contain a therapeutically effective amount of the enzyme(s), preferably in purified form, together with a suitable amount of carrier, so as to provide a form for proper administration to a subject. The formulations should be suitable for the mode of administration.

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

[0364] In some embodiments, the recombinant nepocin polypeptide is provided as a unit dose formulation. By way of non-limiting example, the unit dose can be present in tablets, capsules and others. The unit dose can be in solid, liquid, powder or any other form. The unit dose formulation of the pharmaceutical composition will allow for proper dosing (e.g., based on the amount of immunogenic protein ingested orally) while avoiding potential adverse side effects of over-dosing of the composition.

[0365] In some embodiments, the recombinant nepocin polypeptide or composition thereof, including as a pharmaceutical composition, can be lyophilized from an aqueous solution in the presence of a suitable buffer (e.g., phosphate, citrate, histidine, imidazole buffer) and excipients (e.g., cryoprotective substances such as sucrose, lactose, trehalose, etc.) as needed. The lyophilized cake can be blended with excipients as needed and made into different forms.

[0366] Without being bound by theory, nepocin is thought to be of lower activity or substantially inactive at neutral to basic pH. This can be important in cases where there is a potential for unwanted digestion by the enzyme(s). For example, when the pharmaceutical composition is administered orally, buffering of the composition to a pH of 6.5 or greater can result in a lower activity form of the enzyme(s) such that oral mucosa, esophageal mucosa and other cells that may come into contact with the composition are not digested by the enzyme(s) therein. Similarly, when the composition is added to food, the buffered enzyme(s) will be unable (or have low ability) to digest the food before it is consumed. Under such circumstances, introduction of the composition into the acidic environment of the stomach can result in a decrease in pH and activation of the enzyme(s).

[0367] Thus, in some embodiments, the pharmaceutical composition is buffered to a pH of about 6.5 or higher. In preferred embodiments, the composition is buffered to a pH of about 6.5 to about 8.5. In some embodiments, the composition is in liquid form. In some embodiments, the composition is in semi-solid form. In some embodiments, the composition is in solid form. In some embodiments, the pH of the composition is adjusted in the liquid form and the composition is dried to form a solid. In some embodiments, the enzyme(s) in the composition is / are activated after contact with an acid (i.e., in the stomach).

[0368] In some embodiments, a pharmaceutical composition comprising a recombinant nepocin polypeptide is formulated in a delayed release vehicle such that the enzyme(s) is / are continuously released while the formulation is in the stomach. In some embodiments, the formulation has a pH greater than about 5 prior to contact with 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 acid in the stomach. In preferred embodiments, the formulation is a controlled release formulation. The term "controlled release formulation" includes sustained release and timed release formulations. Controlled release formulations are well known in the art. This includes excipients that allow for sustained, periodic, pulsed or delayed release of a drug. Controlled release formulations include, without limitation, encapsulation of the drug in a matrix; enteric coating; microencapsulation; gels and hydrogels; and any other formulation that allows for controlled release of a drug.

[0369] It should also be understood that in some embodiments, a nepocin polypeptide having proteolytic activity, e.g., a mature nepocin polypeptide having proteolytic activity, can be used in the composition. In some embodiments, the mature nepocin polypeptide having proteolytic activity in the composition is treated to an acidic pH to prepare an active mature polypeptide. In some embodiments, a nepocin polypeptide having proteolytic activity can be used to treat food prior to oral ingestion or can be orally administered simultaneously with oral ingestion of food.

[0370] In some embodiments, the composition is a food product that includes a recombinant neprosin polypeptide. In some embodiments, the food is liquid, semi-solid or solid. In some embodiments, the food product is a bakery product (e.g., cake, muffin, donut, pastry, roll and bread) made from wheat, rye and barley, pasta, cracker, tortilla chips, cereal, etc., that contains gluten or is suspected of containing gluten. In some embodiments, the food product can be consumed with another food product that contains gluten or is suspected of containing gluten. Non-limiting examples of such foods include powder, spread, spray, sauce, dip, whipped cream, candy, chewing gum, syrup, sugar, salt, salad dressing, spice, cheese, butter, margarine, spread, butter, frying shortening, mayonnaise, dairy products, nut butter, seed butter, kernel butter, peanut butter, etc.

[0371] In some embodiments, the recombinant neprosin polypeptide or its composition is mixed with food or used in the pretreatment of food products containing gluten. The recombinant neprosin polypeptide in the food can have enzymatic activity to reduce the level of gluten in the food before or during oral ingestion. In some embodiments, the recombinant neprosin polypeptide is dispersed in the food before consumption at a pH at which it is inactive, such as about 5 or above 5, if necessary. Preferably, the food or additive containing the neprosin polypeptide does not require heating before being orally ingested by the patient so that possible loss of activity of the enzyme(s) due to temperature increase can be minimized.

[0372] In some embodiments, a dispenser containing an internal excipient and an effective amount of 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 the internal excipient are added to the food before the food is consumed. In some embodiments, the internal excipient comprises sodium chloride or sodium iodide or a mixture thereof. In some embodiments, the pharmaceutical composition and / or the internal excipient are in granular form and are sized for efficient dispensing from the dispenser.

[0373] In some embodiments, the recombinant neprosin polypeptide or a composition thereof (e.g., a pharmaceutical composition, an edible composition, or a composition having additional protease, etc.) 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% of neprosin polypeptide. In some embodiments, the amount of recombinant neprosin polypeptide in a composition (such as a pharmaceutical composition or an edible composition) or a 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 in a range between any two of these values (including the endpoints).

[0374] In some embodiments, a composition comprising a pharmaceutical composition comprises a recombinant neprosin polypeptide and one or more additional proteases. In some embodiments, the one or more additional proteases are aspartic proteases, serine proteases, threonine proteases, cysteine proteases, glutamic acid proteases or metalloproteases. In some embodiments, the additional protease is a nepenthes protease such as nepenthesin I and / or nepenthesin II as described herein. In some embodiments, the composition comprises one or more additional exoproteases such as leucine aminopeptidase and carboxypeptidase. In some embodiments, the one or more additional proteases is trypsin. In preferred embodiments, the one or more additional proteases are active at acidic pH (e.g., pH 2-6). Exemplary proteases that may be useful in the present invention include, without limitation, nepenthesin I, nepenthesin II, BACE, cathepsin D, cathepsin E, chymosin (or "rennin"), napsin, pepsin, plasmepsin, presenilin, renin, trypsin, chymotrypsin, elastase and cysteine endoprotease (EP) B2 (also known as EPB2). In preferred embodiments, at least one additional protease is active at an acidic pH such as the pH found in the stomach (e.g., pH 1.5-3.5).

[0375] Combination with Nepenthesis protease In some embodiments, a composition comprising a recombinant nepenthes polypeptide comprises additional proteases that are nepenthesis proteases, such as one or more of nepenthesin I and nepenthesin II. Nepenthesin (EC 3.4.23.12) is a class of aspartic proteases that are present in Nepenthes pitcher leaf secretions, as well as in various 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). Nepenthesin has two known isotypes: nepenthesin I (which is known to have two variants: nepenthesin Ia and nepenthesin Ib) and nepenthesin II. Nepenthesin I mRNA / cDNA sequences and corresponding amino acid sequences have been described from several Nepenthes species, including, inter alia, Nepenthes mirabilis (GenBank accession numbers JX494401 and AFV26024); Nepenthes gracilis (GenBank accession numbers AB114914 and BAD07474) and Nepenthes alata (GenBank accession numbers AB266803 and BAF98915). Nepenthesin II mRNA / cDNA sequences have been described from several Nepenthes species, including, inter alia, Nepenthes mirabilis (GenBank accession numbers JX494402 and AFV26025) and Nepenthes gracilis (GenBank accession numbers AB114915 and BAD07475.1). The sequences are also found in U.S. Patent Application Publication No. 20140186330, which is hereby incorporated by reference in its entirety. Each of the sequences represented by the GenBank accession numbers provided herein is hereby incorporated by reference in its entirety. Exemplary nepenthesin I and nepenthesin II proteases are presented as SEQ ID NOs: 72, 73, 74, 75, 76 and 77.In some embodiments, the biosynthesis of Nepenthes enzyme(s) can be achieved by transforming cells with a vector containing cDNA encoding nepenthesin.

[0376] In some embodiments, nepenthesin is a recombinant nepenthesin 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 nepenthesin I. In some embodiments, nepenthesin comprises the amino acid sequence of nepenthesin I.

[0377] In some embodiments, nepenthesin is a variant of nepenthesin 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 nepenthesin II. In some embodiments, nepenthesin comprises the amino acid sequence of nepenthesin II.

[0378] In some embodiments, nepenthesin I, nepenthesin II, or variants thereof 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 enzymes are synthesized by separate cells and combined in a pharmaceutical composition. In a preferred embodiment, recombinant nepenthesin I, nepenthesin II, or variants thereof are not glycosylated. In some embodiments, recombinant nepenthesin I, nepenthesin II, or variants thereof have a glycosylation pattern different from that of the native enzymes (i.e., nepenthesin I or nepenthesin II isolated from Nepenthes plants). Synthetic (e.g., recombinant) Nepenthes enzyme(s) can be concentrated or purified according to known methods, such as methods for isolating Nepenthes enzyme(s) from plant pitcher fluid.

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

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

[0381] In some embodiments, the ratio of neprosin in the composition to nepenthesin I and / or II is such that the peptidic food antigen is cleaved into sufficiently small and / or harmless fragments so as to be able 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 symptoms of any of these in the intestine of the subject. In some embodiments, the neprosin:nepenthesin ratio is between about 1:100 and about 100:1.

[0382] In some embodiments, nepenthesin I or nepenthesin II in the composition is full-length nepenthesin I or nepenthesin II. In some embodiments, nepenthesin I or nepenthesin II in the composition is the mature form of nepenthesin I or nepenthesin II. In some embodiments, nepenthesin I or nepenthesin II in the composition is the proenzyme of nepenthesin I or nepenthesin II.

[0383] Regarding the proenzyme or propolypeptide of nepenthesin, it is known that nepenthesin I and / or nepenthesin II occur as proenzymes in Nepenthes, and the propolypeptide or proenzyme is cleaved in the pitcher fluid to activate the enzyme. In some embodiments, the composition comprises recombinant neprosin in the form described above, as well as the proenzyme / propeptide of nepenthesin I and / or nepenthesin II. In some embodiments, the propeptide is adjacent to the N-terminus of the enzyme. In some embodiments, the propeptide is the naturally occurring propeptide of the enzyme. In some embodiments, the propeptide is a heterologous propeptide (e.g., derived 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 an 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 for removal of the propeptide and production of 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 more rapidly to speed up the activity of the enzyme in the stomach.

[0384] Use and method The recombinant neprosin polypeptides described herein can be used, alone or as a composition with other proteases, for the degradation of dietary proteins, particularly proteins rich in proline and / or glutamine or other immunogenic proteins, that are not effectively degraded by gastrointestinal enzymes. Degradation of such proteins increases protein absorption and / or decreases immunogenicity, which would have a beneficial effect on intestinal diseases and disorders such as celiac disease, gluten intolerance, irritable bowel syndrome, colitis, Crohn's disease, food allergies and other conditions. Thus, in a further aspect, the present disclosure provides a method for modulating a gluten-intolerance-mediated condition in a subject, the method comprising administering to a subject in need thereof an effective amount of a recombinant neprosin polypeptide described herein. In some embodiments, the condition is celiac disease or wheat allergy.

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

[0386] Neprosin can cleave toxic food peptides into smaller, non-toxic peptides or at least peptides with reduced immunogenicity, either alone or in combination with other proteases such as nepenthesin I and / or nepenthesin II. Together with nepenthesin enzymes, neprosin is active over a wide range of acidic pH, so digestion by this enzyme can be initiated in the acidic environment of the stomach. Administration of neprosin and / or nepenthesin in combination with potentially antigenic food proteins results in a decrease in the immune response to antigenic food proteins after oral ingestion, including a decrease in the infiltration and / or production of intraepithelial lymphocytes in the intestine. Intraepithelial lymphocytes are T cells scattered between the epithelial cells of 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. Administration of recombinant neprosin or its composition, when used in combination with food, can degrade food protein antigens to levels at which the immune response in the intestine, measured by IEL infiltration, is attenuated or eliminated. IEL infiltration due to the presence of peptidic food antigen(s) is an early biological indicator of sensitivity to food antigens (e.g., gluten).

[0387] Accordingly, in one aspect, the use of a recombinant neprosin polypeptide is a method for attenuating or preventing an immune response to a food protein antigen in the intestine of a mammal, the method comprising administering to a subject in need thereof an effective amount of the recombinant neprosin polypeptide described herein. In some embodiments, the recombinant neprosin is administered alone or in combination with nepenthesin I and / or nepenthesin II. In some embodiments, the amount of the pharmaceutical composition is effective to attenuate or prevent intestinal IEL infiltration due to the presence of peptidic food antigen(s).

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

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

[0390] In some embodiments, the use of a recombinant nepocin polypeptide is a method for attenuating or preventing the production and / or recruitment of IELs in the intestine due to the presence of peptidic food antigens in the intestine of a mammal, the method comprising administering to a subject in need thereof an effective amount of the recombinant nepocin polypeptide. In some embodiments, the recombinant nepocin is administered alone or in combination with nepenthesin I and / or nepenthesin II. In some embodiments, the amount of nepocin, alone or in combination with nepenthesin I and / or nepenthesin II, is effective to degrade gluten proteins such that the production and / or recruitment of IELs in the intestine can be attenuated or prevented.

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

[0392] In some embodiments, the use of a recombinant neprosin polypeptide is a method for attenuating or preventing villous atrophy due to the presence of peptidic food antigens in the intestine of a subject, the method comprising administering to the subject in need thereof an effective amount of the recombinant neprosin polypeptide. In some embodiments, the recombinant neprosin is administered alone or in combination with nepenthesin I and / or nepenthesin II. In some embodiments, the amount of neprosin, alone or in combination with nepenthesin I and / or nepenthesin II, is effective to degrade gluten protein such that villous atrophy in the intestine can be attenuated or prevented. In some embodiments, the villous atrophy is a result of intestinal inflammation.

[0393] In some embodiments, the use of a recombinant neprosin polypeptide is a method for treating and / or alleviating 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 the subject in need thereof an effective amount of the recombinant neprosin polypeptide for treating and / or alleviating at least one symptom associated with an immune response to the presence of gluten or other antigenic proteins. In some embodiments, such symptoms include, without limitation, "foggy mind", depression, anxiety, ADHD-like behavior, abdominal pain, abdominal bloating, diarrhea, constipation, headache, migraine, bone or joint pain, chronic fatigue, small intestine damage, generation of tissue transglutaminase (tTG) antibodies, severe acne, vomiting, weight loss, irritability, iron deficiency anemia, arthritis, pins and needles sensation in the limbs, infertility and stomatitis. In some embodiments, the recombinant neprosin is administered alone or in combination with nepenthesin I and / or nepenthesin II.

[0394] In some embodiments, the use of the recombinant nepocin polypeptide is a method for attenuating or preventing the symptoms of celiac disease resulting from the presence of partially hydrolyzed gluten or wheat protein in the intestine of a patient having celiac disease, the method comprising administering to the patient an effective amount of the recombinant nepocin polypeptide described herein. In some embodiments, the recombinant nepocin is administered alone or in combination with nepenthesin I and / or nepenthesin II. In some embodiments, the amount of nepocin, alone or in combination with nepenthesin I and / or nepenthesin II, is effective to attenuate or prevent the symptoms of celiac disease.

[0395] In another aspect, there is provided 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, the method comprising treating a food containing gluten or suspected of containing gluten with an effective amount of the recombinant nepocin polypeptide or a composition thereof prior to consumption by the patient. In some embodiments, the food is combined with an effective amount of the recombinant nepocin polypeptide or a composition thereof during its preparation. In some embodiments, the nepocin polypeptide is added after any heating step in food preparation. In some embodiments, the nepocin polypeptide is added before one or more heating steps in food preparation. In some embodiments, the recombinant nepocin is administered alone or in combination with nepenthesin I and / or nepenthesin II.

[0396] In some embodiments, the use of the recombinant neprosin polypeptide is a method for improving the digestibility of proteins from food in a subject having an intestinal disorder, the method comprising administering to the subject in need thereof an effective amount of the recombinant neprosin polypeptide. In some embodiments, the recombinant neprosin is administered alone or in combination with nepenthesin I and / or nepenthesin II. In some embodiments, the amount of neprosin, alone or in combination with nepenthesin I and / or nepenthesin II, is effective to break down proteins sufficiently to improve protein absorption 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, the recombinant neprosin or a composition thereof is administered to the subject prior to oral ingestion of a potentially antigenic food or protein. In some embodiments, the recombinant neprosin or a composition thereof is administered to the subject concurrently with oral ingestion of a potentially antigenic food or protein. In some embodiments, the recombinant neprosin or a composition thereof is administered to the subject after oral ingestion of a potentially antigenic food or protein. In some embodiments, the recombinant neprosin or a composition thereof is administered to the subject immediately after oral ingestion of a potentially antigenic food or protein. In some embodiments, the recombinant neprosin or a composition thereof is administered to the 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 a food allergy. In some embodiments, gut antigen protein sensitivity correlates directly or indirectly with attention deficit hyperactivity disorder, autism, rheumatoid arthritis, fibromyalgia, and / or dermatitis herpetiformis. It is further contemplated that removal of such antigenic gut proteins from the gut using the compositions of the invention 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 orally administered before, during, or immediately after consumption of gluten-containing or other immunogenic foods.

[0400] In some embodiments, the recombinant neprosin polypeptide or composition thereof is administered to the subject prior to oral ingestion by the subject of foods suspected of containing gluten or containing gluten. In some embodiments, the neprosin polypeptide or composition thereof is administered during a period in which the enzyme is at least partially effective (e.g., at least about 10%, 20%, 50%, 70%, 90% of its native activity) in the degradation of gluten in the food that the subject is to orally ingest. In some embodiments, the neprosin polypeptide or composition thereof is administered within about 4 hours, 3 hours, 2 hours, 1 hour, or 30 minutes prior to oral ingestion of food by the subject.

[0401] In some embodiments, the neprosin polypeptide or composition thereof is administered to the subject simultaneously with oral ingestion by the subject of potentially immunogenic foods. In some embodiments, the neprosin polypeptide or composition thereof is administered with the food. In some embodiments, the neprosin polypeptide or composition thereof is administered separately from the food.

[0402] In some embodiments, the neprosin polypeptide or a composition thereof is administered to a subject immediately following oral ingestion by a subject of a potentially immunogenic food. In some embodiments, the neprosin polypeptide or a composition thereof is administered during a period in which at least a portion (e.g., at least about 10%, 20%, 50%, 70%, 90%) of the antigen(s) in the food is still in the subject's stomach. In some embodiments, the neprosin polypeptide or a composition thereof is administered within 4 hours, 3 hours, 2 hours, 1 hour, or 30 minutes following oral ingestion of food by the subject.

[0403] Generally, the neprosin polypeptide or a composition thereof is administered in an amount sufficient to produce the desired effect of detoxifying the safe and peptidic food antigen(s). The dosage of the neprosin polypeptide or a composition thereof can vary depending on factors such as the specific enzyme or combination administered, the subject's sensitivity to the food, the amount and type of antigen-containing food orally ingested, the pharmacodynamic properties of the enzyme, the mechanism of administration, the age, health, and weight of the recipient, the nature and extent of the symptoms, the frequency of treatment, and the type of any concurrent treatment, if any, as well as the clearance rate of the enzyme. One of ordinary skill in the art can determine an appropriate dosage based on the above factors. The neprosin polypeptide or a composition thereof can be initially administered at a suitable dosage that can be adjusted as needed depending on the clinical response. In vitro assays can be used as needed to assist in identifying the optimal dosage range. The exact dosage to be used in the formulation can 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 an adult subject can be proportionally adjusted for pediatric and infant subjects and can also be adjusted for other administrations or other formats, e.g., proportionally to the molecular weight or immune response. The administration or treatment can be repeated at appropriate intervals at the discretion of the physician.

[0405] In some embodiments, the effective amount of the recombinant neprosin polypeptide or composition thereof is between about 1 mg and about 1 g. The dosage is less than about 5 mg per 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 (including the endpoints), and sub-ranges between any two of these values.

[0406] In some embodiments, the recombinant neprosin polypeptide is administered at an enzyme amount of about 0.001 mg to about 1000 mg per kg of body weight per day, or at a dosage of about 1 mg to about 100 g per dosage for an average person. In some embodiments, the recombinant neprosin polypeptide can be administered at 0.001, 0.01, 0.1, 1, 5, 10, 50, 100, 500 or 1000 mg / kg body weight per day, and in ranges (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 per dosage, and in ranges (including endpoints) between any two of these values. 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 orally ingests a food containing the antigenic protein and / or how much of such food is consumed. The amounts of the recombinant neprosin polypeptide listed herein can relate to the total enzyme or each enzyme in the composition.

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

[0408] In preferred 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 10:1 and about 100:1.

[0409] In some embodiments, the ratio of the substrate (total protein) to the enzyme (neprosin or a combination of Nepenthes enzymes) administered is between about 1:1 and about 15,000:1. In some embodiments, the ratio of the substrate to the enzyme is between about 10:1 and about 15,000:1. In some embodiments, the ratio of the substrate to the enzyme is between about 100:1 and about 15,000:1. In some embodiments, the ratio of the substrate to the enzyme is between about 500:1 and about 15,000:1. In some embodiments, the ratio of the substrate to the enzyme is between about 1000:1 and about 15,000:1. In some embodiments, the ratio of the substrate to the enzyme is between about 5000:1 and about 15,000:1. In some embodiments, the ratio of the substrate to the enzyme is between about 10,000:1 and about 15,000:1. In some embodiments, the ratio of the substrate to the enzyme is between about 1:1 and about 10,000:1. In some embodiments, the ratio of the substrate to the enzyme is between about 10:1 and about 10,000:1. In some embodiments, the ratio of the substrate to the enzyme is between about 100:1 and about 10,000:1. In some embodiments, the ratio of the substrate to the enzyme is between about 500:1 and about 10,000:1. In some embodiments, the ratio of the substrate to the enzyme is between about 1000:1 and about 10,000:1. In some embodiments, the ratio of the substrate to the enzyme is between about 5000:1 and about 10,000:1. In some embodiments, the ratio of the substrate to the enzyme is between about 1:1 and about 5000:1. In some embodiments, the ratio of the substrate to the enzyme is between about 10:1 and about 5000:1. In some embodiments, the ratio of the substrate to the enzyme is between about 100:1 and about 5000:1. In some embodiments, the ratio of the substrate to the enzyme is between about 500:1 and about 5000:1. In some embodiments, the ratio of the substrate to the enzyme is between about 1000:1 and about 5000:1. In some embodiments, the ratio of the substrate to the enzyme is between about 1:1 and about 1000:1. In some embodiments, the ratio of the substrate to the enzyme is between about 10:1 and about 1000:1. In some embodiments, the ratio of the substrate to the enzyme is between about 100:1 and about 1000:1. In some embodiments, the ratio of the substrate to the 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. This includes any value (including endpoints) within any of these ranges, and sub-ranges between any two of these values.

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

[0411] In some embodiments, the pharmaceutical composition is administered regardless of whether the patient has orally ingested (e.g., deliberately orally ingested) a food containing a potentially immunogenic protein. In some embodiments, the pharmaceutical composition is administered on an as-needed basis, e.g., before, during, and / or after a meal that may be contaminated with a potentially immunogenic protein or of unknown potentially immunogenic protein content. In some embodiments, the pharmaceutical composition is administered on a regular basis. In some embodiments, the pharmaceutical composition is administered at least once per day. In some embodiments, the pharmaceutical composition is administered 2, 3, 4 times or more per day. In some embodiments, the pharmaceutical composition is administered in conjunction with each meal and / or snack (e.g., before, during, or after). In some embodiments, the pharmaceutical composition is included as part of a sustained release formulation with continuous release of the enzyme(s) to allow for intermittent snacking, etc., regardless of the antigenic protein content of the food.

[0412] In some embodiments, also as described herein, the recombinant nepocin polypeptide can be administered as the sole active agent or in combination with other agents, including other compounds that have demonstrated the same or similar therapeutic activity and have been determined to be safe and effective for such combined administration (simultaneously, sequentially or separately, or by co-formulation). In some embodiments, as described above, the pharmaceutical composition is administered with additional enzymes, such as gastric proteases, aspartic proteases (such as pepsin, pepsinogen or those described by Chen et al., Gene, 2009, 442:108-118), and enzymes such as another prolyl endopeptidase (PEP), dipeptidyl peptidase IV (DPP IV) and dipeptidyl carboxypeptidase (DCP) or cysteine protease B (see, for example, U.S. Patent No. 7,910,541). In some embodiments, the additional enzyme is administered in the form of bacteria that produce and / or secrete the additional enzyme. In some embodiments, the bacteria are engineered to produce and / or secrete nepocin alone or in combination with nepentesin I and / or nepentesin 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 co-administered with the pharmaceutical composition include inhibitors of tissue transglutaminase, anti-inflammatory agents such as amylase, glucoamylase, endopeptidase, 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 chromoglycate (chromolyn), pemirolast, proxichromil, repirinast, doxantrazole, anlelexanox, nedocromil, and probicromil, anti-ulcer agents, anti-allergy agents such as antihistamines (e.g., acrivastine, cetirizine, desloratadine, ebastine, fexofenadine, levocetirizine, loratadine, and mizolastine), inhibitors of transglutaminase 2 (TG2), anti-TNFα agents, as well as antibiotics. In some embodiments, the additional agent is a probiotic. Probiotics include, without limitation, species and strains of Lactobacillus, yeast, Bacillus, or Bifidobacterium. In some embodiments, the other agent is an elastin. In some embodiments, the other agent is administered in the form of bacteria that produce and / or secrete the additional agent.

Example

[0414] The following examples, including the experiments and the results achieved, are provided for illustrative purposes only and should not be construed as limiting the invention. (Example 1) Expression of Neprosin in Mammalian Cells

[0415] On the day of transfection, the viable cell density and percent viability of the prepared cells were determined. Using pre-warmed Expi293™ Expression Medium, the cells were diluted to a final viable cell density of 3×106 viable cells / mL, and the percent viability should be ≧95%. The culture flask was gently swirled to mix the cells and prepare them for transfection. Transient transfection was performed using the recommended protocol of the ExpiFectamine™ 293 Transfection Kit. The viable cell density and percent viability were tested every two days. If the viable cell density was less than half of the peak maximum value or the viable cell percent viability was below 70%, the culture was harvested. Neprosin I (Npr1) was secreted into the medium, and the supernatant was collected on day 5 after transfection. The entire culture was centrifuged in a tabletop centrifuge at 4,000 RCF for 30 minutes in a 50 mL Falcon tube, and the filtered supernatant was filtered using a 0.22 μm filter. Selected samples were subjected to purification.

Table 1

[0416] Formation of Active Neprosin Protease Polypeptide An 80 μL aliquot of approximately 40 μM Expi293 or ExpiCHO purified Npr1 (mSP-His-Npr1; prepared from cells expressing SEQ ID NO: 40) was added to a 3.5K MWCO Slide-a-lyzer MINI dialysis device (Thermo Scientific, USA) containing 14 mL of 100 mM glycine, pH 2.5. The dialysis device was incubated at 37 °C and 250 rpm. At the time of incubation, 4 μL of the enzyme was removed and diluted 1:5 in 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 minutes and 10 μL was loaded onto a 12-well NuPAGE gel (4-12% Bis-Tris). SDS-PAGE was run at 200 V for 35 minutes and the gel was stained with Coomassie blue. The results are shown in Figure 2.

[0417] pH activation profile of neprosin protease polypeptide A 50 μL aliquot of approximately 8 μM purified Npr1 (mSP-His-Npr1; SEQ ID NO: 40) was added to a 3.5K MWCO Slide-a-lyzer MINI dialysis device containing 14 mL of 1× MacIlvaine buffer at the designated pH (i.e., pH 2.5, 3, 3.5, 4, 4.5 or 5), 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. The dialysis device was incubated at 37 °C and 250 rpm. At 60 minutes, 50 μL of the enzyme sample was recovered and placed in a PCR tube. 10 μL of these samples was immediately mixed 1:1 with 2× SDS-PAGE sample buffer. The remaining samples were left at RT for 5 hours without shaking and then mixed with sample buffer as described above. The sample in SDS-PAGE buffer was heated at 95 °C for 10 minutes and 10 μL was loaded onto a 12-well NuPAGE gel (4-12% Bis-Tris). SDS-PAGE was run at 200 V for 35 minutes and the gel was stained with Coomassie blue. The results are shown in Figure 3.

[0418] Activity of activated neprosin protease in gliadin substrate An approximately 1.2 mg / mL solution of solubilized gliadin stock (previously prepared and frozen) was centrifuged at 15,000 rpm and the soluble fraction was transferred to a new tube. The soluble fraction was diluted 1:10 in ddH2O to obtain an approximately 0.12 mg / mL working stock. A 50 μL aliquot of approximately 0.12 mg / mL gliadin was added to 40 μL of 100 mM sodium acetate buffer, pH 4, in a PCR tube. An aliquot of neprosin enzyme, 10 μL of 0.06 mg / mL or 0.006 mg / mL enzyme preparation, was added to initiate the reaction and incubated at 37 °C, 900 rpm for 5 or 30 minutes. The reaction tubes were placed in a preheated (95 °C) thermocycler and the reaction was quenched by heating at 95 °C for 10 minutes. Samples were diluted 15,000× before analysis using RIDASCREEN® Gliadin Competitive ELISA. Samples without enzyme were used to calculate the percentage of gliadin remaining. The results are shown in Figure 4. The neprosin preparation activated by treatment at acidic pH exhibits significantly greater proteolytic activity compared to the non-activated neprosin polypeptide prepared from cells expressing mSP-His-Npr1. (Example 3) Transfection and expression by neprosin in Pichia cells

[0419] Transfection of Pichia cells with the neprosin gene 200 ng of linearized Pme1 DNA was added to 30 μL of Pichia BG10 electrocompetent cells and gently mixed. The cell and DNA mix was transferred to a chilled 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: 1 M sorbitol, 1:1 (v / v)) was added to the cuvette, gently mixed with the cell solution, and transferred to different tubes. The electroporated cells were incubated at 20 °C for 3 h at 100 rpm. Next, 100 μL of the incubated cells were spread on YPD + 800 μg / mL G418 agar plates at 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 for 48 h at 250 rpm. The cells were stored at -80 °C in 25% glycerol. Expression of Neprosin in Pichia Using a glycerol stock of Pichia cells containing the neprosin (Npr1) gene, streaks were made on YPD + 800 μg / mL G418 agar plates. The plates were incubated at 30 °C for 48 h. Single colonies obtained from the agar plates were used to inoculate 50 mL of BMGY. This starter culture was incubated at 30 °C for 24 h at 200 rpm and then used to inoculate a shaking flask containing BMGY (1:50). The culture was grown at 30 °C for 48 h at 250 rpm. The cells were collected by centrifugation and then resuspended in BMY medium (BMMY) containing 2% methanol to induce protein expression. Secreted Npr1 was obtained by collecting the supernatant after 24 h (4,000 rpm, 20 min). The supernatant was filtered using a 0.45 μm filter and subjected to purification. SDS-PAGE analysis of WT, N216A, and N216A, N324A Npr1 (SEQ ID 56, 60, and 66, respectively) is shown in Figure 5.

Table 2

[0420] Formation of active neprosin protease polypeptide WT Npr1, N216A Npr1 or N216A / N324A Npr1 (SEQ ID Nos. 54, 60 and 66, respectively) prepared from Pichia cells was added to a 10K MWCO Slide-A-Lyzer dialysis cassette (Thermo Scientific, USA) and dialyzed against 100 mM glycine, pH 2.5 at 37 °C for 4 h with gentle stirring. Samples of the dialyzed protein were 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 Fig. 6. Activated WT neprosin appears as two glycosylated polypeptides around 29 and 35 kDa. Activated N216A neprosin and N216A / N324A neprosin appear as a single glycosylated species around 29 kDa.

[0421] N-terminal sequencing of the activated WT neprosin band using in-gel digestion LC-MS / MS analysis (Thermo Orbitrap Exploris 240) identified P199-S200 (SEQ ID No. 54) as the cleavage site between the propeptide and activated Npr1. This analysis also confirmed the identity of both protein bands as activated Npr1, and the difference in their molecular weights is due to differences in mannose chain length glycosylation.

[0422] Activity of activated...

Claims

[Claim 1] The invention described herein.

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