Engineered proteases with enhanced autolysis resistance

Abstract

The present disclosure relates to engineered proteases, including trypsin, a Lys-C protease, and an Asp-N protease, which have enhanced autolysis resistance. Also disclosed herein are methods of using such engineered enzymes to improve the detection of a target analyte protein in an analytical assay.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of and priority to U.S. Provisional Patent Application No. 63 / 395,986, filed on August 8, 2022. The entire disclosure of that application is hereby incorporated by reference herein.

[0003] Reference to Sequence Listing XML

[0004] This patent application contains a sequence listing that has been submitted electronically in XML format. The sequence listing XML is incorporated herein by reference. The XML file created on August 1, 2023 is named WAC-396WO_SL.xml and is 12,004 bytes in size. Background Art

[0005] Proteases are used in a variety of analytical applications. For example, proteases are used for sequencing or peptide mapping of proteins and quality control testing of therapeutic proteins such as monoclonal antibodies, antibody-drug conjugates (ADCs) and enzyme replacement therapy (ERT). These analyses are usually performed with liquid chromatography-mass spectrometry (LC-MS) instruments. However, there are problems in obtaining quality data when proteases act on themselves through a process called autolysis, which produces undesirable peptide byproducts of proteases that can mask the detection peaks of related protein analytes. This contaminates samples with non-informative and destructive peptides. Therefore, there is a need for compositions and methods that minimize protease autolysis. Summary of the invention

[0006] The present disclosure relates to recombinant proteases, such as endopeptidase Lys-C (Lys-C), Asp-N protease (Asp-N) and trypsin, containing one or more conservative amino acid substitutions that confer enhanced autolysis resistance to the enzyme. Also disclosed are methods of using such enzymes in analytical assays (e.g., liquid chromatography-mass spectrometry (LC-MS), etc.) to improve detection and analysis of target protein analytes.

[0007] In one aspect, the present disclosure provides a recombinant protease comprising one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more) conservative amino acid substitutions that enhance the autolysis resistance of the protease compared to the autolysis resistance of the protease in the absence of the one or more conservative substitutions, wherein the protease is Lys-C.

[0008] In one aspect, the disclosure provides a recombinant protease comprising one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more) conservative amino acid substitutions that enhance the autolysis resistance of the protease compared to the autolysis resistance of the protease in the absence of the one or more conservative substitutions, wherein the protease is Asp-N.

[0009] In one aspect, the disclosure provides a recombinant protease comprising one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more) conservative amino acid substitutions that enhance the autolysis resistance of the protease compared to the autolysis resistance of the protease in the absence of the one or more conservative substitutions, wherein the protease is trypsin.

[0010] In some embodiments, one or more conservative substitutions include at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, or at least 15 conservative substitutions. Alternatively or in addition, one or more conservative amino acid substitutions include 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 conservative substitutions. Alternatively or in addition, one or more conservative substitutions are lysine (Lys) to arginine (Arg) substitutions. Alternatively or in addition, one or more conservative substitutions include substitutions of Lys to Arg at one or more amino acid residues selected from residues 2, 39, 52, 54, 62, 104, 173, 178, 183, 205, 235, 254, 311, 360 and 408 of SEQ ID NO: 1 or residues 30, 49, 106, 155 and 203 of SEQ ID NO: 2. Alternatively or in addition, at least one or more conservative substitutions are substitutions of aspartic acid (Asp) to glutamic acid (Glu). Alternatively or in addition, one or more conservative substitutions include substitutions of Asp to Glu at one or more amino acid residues selected from residues 2, 14, 46 and 130 of SEQ ID NO: 5. Alternatively or in addition, one or more conservative substitutions also include substitutions of Asp to Glu at one or more amino acid residues 67 and 71 of SEQ ID NO: 5. Alternatively or in addition, one or more conservative substitutions are substitutions of Arg to Lys. Alternatively or additionally, the one or more conservative substitutions include an Arg to Lys substitution at one or more amino acid residues selected from residues 45, 49, 99 and 107 of SEQ ID NO:8.

[0011] In some embodiments, the autolysis resistance of the protease is enhanced by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% compared to the autolysis resistance of the protease in the absence of one or more conservative substitutions.

[0012] In some embodiments, the protease comprises one or more chemical modifications. Alternatively or in addition, the one or more chemical modifications include an alkyl moiety, an acetyl moiety, an amide moiety, an ester moiety, an imine moiety, an amidino moiety, a guanidino moiety, or a thioether moiety. Alternatively or in addition, the alkyl moiety is selected from a methyl moiety, a dimethyl moiety, an octanal moiety, and a cyclodextrin monoaldehyde moiety.

[0013] In some embodiments, the protease is porcine, bovine, rat, murine, avian, human, bacterial, fungal, or plant. In some embodiments, the protease is artificial or synthetic.

[0014] In some embodiments, the active site of the protease contains no amino acid substitutions.

[0015] In some embodiments, the protease comprises the amino acid sequence of SEQ ID NO: 3 or SEQ ID NO: 4. Alternatively or additionally, the protease comprises the amino acid sequence of SEQ ID NO: 6 or SEQ ID NO: 7. Alternatively or additionally, the protease comprises the amino acid sequence of SEQ ID NO: 9.

[0016] In one aspect, the present disclosure provides a method for reducing the level of peptide byproducts of protease autolysis in an analytical assay, the method comprising using a recombinant protease of any of the aforementioned aspects and embodiments. Alternatively or in addition thereto, the analytical assay is selected from liquid chromatography (LC), LC-mass spectrometry (LC-MS), LC-UV, capillary electrophoresis (CE), gel electrophoresis (GE) and matrix-assisted laser desorption / ionization (MALDI). Alternatively or in addition thereto, the analytical assay is LC-MS.

[0017] In one aspect, the present disclosure provides polynucleotides (e.g., DNA or RNA) comprising a nucleic acid sequence encoding a recombinant protease of any of the aforementioned aspects and embodiments. In some embodiments, the polynucleotide encodes a polypeptide or a variant thereof having an amino acid of SEQ ID NO: 3 or SEQ ID NO: 4, which has at least 85% (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher) sequence identity with SEQ ID NO: 3 or SEQ ID NO: 4. In some embodiments, the polynucleotide encodes a polypeptide having the amino acids of SEQ ID NO: 6 or SEQ ID NO: 7, or a variant thereof, which has at least 85% (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence identity to SEQ ID NO: 6 or SEQ ID NO: 7. In some embodiments, the polynucleotide encodes a polypeptide having the amino acids of SEQ ID NO: X, or a variant thereof, which has at least 85% (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence identity to SEQ ID NO: 9. In some embodiments, the polynucleotide further comprises a nucleic acid sequence encoding a protein tag (eg, a His-tag, such as a 6X-His-tag (SEQ ID NO: 10)).

[0018] In one aspect, the disclosure provides nucleic acid expression vectors comprising the polynucleotides of the aforementioned aspects and embodiments. In some embodiments, the polynucleotides are operably connected to a promoter sequence (e.g., a mammalian promoter, a bacterial promoter, a fungal promoter, or an insect promoter). In some embodiments, the expression vector also comprises one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8 or more) regulatory sequences selected from 5' untranslated region (UTR), 3'UTR, enhancer, insulator, intron, RNA export element, polyadenylation signal, and transcription terminator. In some embodiments, the expression vector is a plasmid. In some embodiments, the expression vector is a viral vector.

[0019] In one aspect, the present disclosure provides a host cell comprising a recombinant protease, polynucleotide or expression vector of any of the aforementioned aspects and embodiments. In some embodiments, the host cell is a mammalian cell. In some embodiments, the host cell is a bacterial cell. In some embodiments, the host cell is a fungal cell. In some embodiments, the host cell is an insect cell. In some embodiments, the host is a plant cell.

[0020] In one aspect, the present disclosure provides a kit comprising the recombinant protease, polynucleotide, expression vector, or host cell of any of the preceding aspects and embodiments.

[0021] definition

[0022] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those of ordinary skill in the art of the subject matter claimed. Generally speaking, the nomenclature and techniques used in connection with cell and tissue culture, molecular biology, and protein and polynucleotide chemistry as described herein are those well known and commonly used in the art. It should be understood that the general description above and the detailed description below are only exemplary and illustrative, and do not limit any subject matter claimed. The section titles used herein are only for organizational purposes and should not be construed as limiting the subject matter.

[0023] As used herein, the phrase "analytical assay" refers to any known assay in the relevant art for analyzing proteins. Non-limiting examples of analytical assays include those for extraction, separation, detection, sequence analysis, structural analysis, post-translational modification analysis, and target protein ("target analyte") functional assessment, etc. Specific examples of analytical assays include liquid chromatography (LC), LC-mass spectrometry (LC-MS), LC-UV, capillary electrophoresis (CE), gel electrophoresis (GE), matrix-assisted laser desorption / ionization (MALDI), hydrogen-deuterium exchange, protein sequencing, peptide mapping by electrophoresis, Western blotting, protein nuclear magnetic resonance (NMR), protein footprinting, affinity purification, protein conformation studies, and proteomics, etc.

[0024] As used herein, the phrase "autolysis resistance" or its variants refers to the property of the recombinant proteases described herein (e.g., Lys-C, Asp-N and trypsin) to resist proteolytic cleavage (i.e., self-cleavage) via their own active sites. Protease itself is a protein containing amino acid residues that can serve as substrate residues for the active site of the protease. Therefore, replacing these residues with conservative amino acid residues that are not natural substrates for the active site of the enzyme and / or chemically modifying the residues (e.g., alkylation) can enhance the resistance of the protease to self-cleavage. The recombinant protease disclosed herein may contain any number of amino acid residues (e.g., 1, 2, 3, 4, 5, 6 or more amino acid residues) that are used as substrates for the protease domain of the enzyme. Therefore, the autolysis resistance of the protease to different degrees can be conferred by conservative amino acid substitutions at these residues. For example, the autolysis resistance of the protease can be conferred by 1, 2, 3, 4, 5, 6 or more conservative amino acid substitutions. The enhancement of autolysis resistance can be any amount, including, for example, an enhancement of the autolysis resistance of the protease by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% compared to the autolysis resistance of the protease in the absence of the conservative substitution. In some embodiments, the degree of autolysis resistance of the protease is proportional to the number of conservative amino acid substitutions (i.e., more conservative amino acid substitutions result in greater autolysis resistance).

[0025] As used herein, the phrase "chemical modification" refers to any process that can transform a molecule or macromolecule by a chemical reaction or a series of chemical reactions. Non-limiting examples of chemical modifications include the addition of an alkyl moiety, an acetyl moiety, an amide moiety, an ester moiety, an imine moiety, an amidino moiety, a guanidino moiety, or a thioether moiety. Non-limiting examples of alkyl moieties include methyl moieties, dimethyl moieties, octanal, and cyclodextrin monoaldehyde moieties.

[0026] As used herein, the terms "conservative substitution", "conservative amino acid substitution" and "conservative mutation" refer to substitution of one or more amino acids by one or more different amino acids that exhibit similar physicochemical properties, such as polarity, electrostatic charge and steric bulk. These properties for each of the twenty naturally occurring amino acids are summarized in Table 1 below.

[0027] Table 1. Representative physicochemical properties of naturally occurring amino acids

[0028]

[0029]

[0030] As can be seen from the table, the conservative amino acid family includes (i) G, A, V, L and I; (ii) D and E; (iii) C, S and T; (iv) H, K and R; (v) N and Q; and (vi) F, Y and W. Therefore, a conservative mutation or substitution is a mutation or substitution that replaces a member of the same amino acid family with an amino acid (e.g., replacing Thr with Ser or replacing Arg with Lys).

[0031] As used herein, the phrase "peptide byproducts of protease autolysis" refers to protease fragments (e.g., Lys-C, Asp-N, or trypsin) produced by protease autolysis. Peptide byproducts of protease autolysis may have different sizes, depending on the length of the protease amino acid sequence and the number of amino acid residues contained therein that may serve as substrates for proteolytic cleavage of the active site of the protease. Such peptide byproducts are generally undesirable in the context of certain protein assays (e.g., analytical assays disclosed herein) because they may interfere and negatively affect the sensitivity and / or specificity of the measurements produced by these assays. 43,44,45,46,47,48,49,50,55,65,70,75,80,85,90,91,92,93,94,100,100,100,100,100,100,100,100,100,100,100,100,100,100,100,100,100,100,100,100,100,100,100,100,100,100,100,100,100,100,100,100,100,100,100,100,100,100,100,100,100,100,100,100,100,100,100,100,100 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830, 840, 850, 860, 870, 880, 890, 900

[0032] " Percentage of sequence identity (%) " about reference polynucleotide or peptide sequence is defined as after comparing sequence and introducing breach to realize maximum sequence identity percentage when necessary, the percentage of nucleic acid or amino acid identical with the nucleic acid or amino acid in reference polynucleotide or peptide sequence in candidate sequence.Comparison for measuring percentage nucleic acid or amino acid sequence identity purpose can be realized in a variety of ways within the capabilities of those skilled in the art, for example, using computer software such as BLAST, BLAST-2 or Megalign software available to the public.Those skilled in the art can determine the appropriate parameters for comparing sequence, including any algorithm required for realizing maximum comparison on the total length of the sequence compared.For example, sequence comparison computer program BLAST can be used to produce sequence identity percentage numerical value.As an illustration, given nucleic acid or amino acid sequence A to given nucleic acid or amino acid sequence B, with given nucleic acid or amino acid sequence B or with respect to given nucleic acid or amino acid sequence B sequence identity percentage (alternatively, can be expressed as given nucleic acid or amino acid sequence A to given nucleic acid or amino acid sequence B, with given nucleic acid or amino acid sequence B or with respect to given nucleic acid or amino acid sequence B has certain sequence identity percentage) is calculated as follows:

[0033] 100 times (fraction of X / Y)

[0034] wherein X is the number of nucleotides or amino acids that are scored as identical matches in an alignment of A and B by a sequence alignment program (e.g., BLAST), and wherein Y is the total number of nucleic acids in B. It will be understood that when the length of nucleic acid or amino acid sequence A is not equal to the length of nucleic acid or amino acid sequence B, the percent sequence identity of A to B will not be equal to the percent sequence identity of B to A.

[0035] As used herein, the term "recombinant" refers to a protein encoded by a nucleic acid that has been cloned into an expression system that is capable of transcribing a gene for translation into a protein. Genetic modification of recombinant nucleic acids can be used to produce mutant proteins, such as mutant proteases having one or more (e.g., 1, 2, 3, 4, 5, 6 or more) conservative amino acid substitutions.

[0036] As used herein, the term "vector" includes nucleic acid vectors, such as DNA vectors such as plasmids, RNA vectors, viruses or other suitable replicons (e.g., viral vectors). A variety of vectors have been developed for delivering polynucleotides encoding exogenous proteins to prokaryotic or eukaryotic cells. Expression vectors suitable for the compositions and methods described herein contain polynucleotide sequences and, for example, other sequence elements for expressing proteins and optionally integrating these polynucleotide sequences into the genome of host cells. As described herein, certain vectors that can be used to express one or more (e.g., 1, 2, 3 or more) recombinant proteases include plasmids containing regulatory sequences (such as promoters and enhancer regions) that direct gene transcription. Other useful vectors for expressing proteases contain polynucleotide sequences that enhance the translation rate of these genes or improve the stability or nuclear export of mRNA produced by gene transcription. These sequence elements may include, for example, 5' and 3' untranslated regions (UTRs), internal ribosome entry sites (IRESs), and polyadenylation signal sites to guide the efficient transcription of genes carried on the expression vector. Expression vectors suitable for the compositions and methods described herein may also contain polynucleotides encoding markers that are used to select cells containing such vectors. Examples of suitable markers are genes encoding resistance to antibiotics such as ampicillin, chloramphenicol, kanamycin, nourseothricin, bleomycin, neorseothricin, carbenicillin, tetracycline, streptomycin and spectinomycin. DETAILED DESCRIPTION

[0037] The present disclosure is characterized in that engineered proteases, including endopeptidase Lys-C (Lys-C), Asp-N protease (Asp-N) and trypsin, exhibit enhanced resistance to autolysis, i.e., resistance to self-cleavage. Enhanced resistance to autolysis is generated by incorporating one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 or more) conservative amino acid substitutions at specific amino acid residues of the protease, i.e., those residues serve as natural substrates for the active site of the protease itself. Such autolysis-resistant proteases can be used for analytical assays that are expected to minimize interference from protease autolysis byproducts, such as liquid chromatography-mass spectrometry (LC-MS) and the like.

[0038] Protease autolysis interferes and negatively affects the sensitivity and specificity of the measurement based on LC-MS. A problem encountered during peptide mapping analysis etc. is that protease itself is protein, and will self-digest (" autolysis ") into peptide by-products. When two or more proteases are used to the same sample, the problem of being derived from the autolysis background peptide becomes more significant. Therefore, it is expected that the digestion mixture only with the peptide fragments from the protein analyte of interest (" target analyte ") will be polluted by the peptides from the protease used in the sample preparation. The chromatographic peaks of these protease fragments occur in the separation process based on high performance liquid chromatography (HPLC), therefore making the identification and structural characterization of the target analyte protein more difficult.

[0039] To date, it has been desirable to minimize autolysis reaction byproducts in LC-MS analysis in order to reduce byproduct interference during detection of target analytes. Previous studies have shown that incorporating non-conservative amino acid substitutions into rodent trypsin enhances autolysis resistance ( Biochem Biophys Res Commun. 243:56-60, 1998; the entire text of which is incorporated herein by reference); however, because non-conservative substitutions can allow the introduction of amino acids with different physicochemical properties than the substituted amino acids, such substitutions are generally undesirable because they have the potential to disrupt the tertiary structure of the protein and thus its function. Here, the present disclosure provides proteases that have been genetically engineered to introduce one or more conservative amino acid substitutions and are optionally chemically or enzymatically alkylated to enhance resistance to autolysis.

[0040] Recombinant autolysis-resistant protease

[0041] Lys-C

[0042] Lys-C (30 kDa) is a bacterial serine protease that hydrolyzes peptide bonds on the carboxyl side of lysine (Lys) residues, particularly Lys residues followed by a proline residue. The enzyme generally produces long peptide fragments of low complexity. Lys-C exhibits optimal protease activity in the pH range of 7.0-9.0 and is highly resistant to strong denaturing conditions (e.g., high concentrations of urea). Lys-C is naturally present in Lysobacter spp., including, for example, Lysobacter enzymogenes (Jekel et al., Anal Biochem 134:347-54, 1983), Lysobacter antibiotus, Lysobacter sp. Root96, Lysobacter maris, as well as Shewanella spp., Aquimonas, Pseudofulvimonas, Tahibacter, Thalassocella, Achromobacter lyticus (M497-1) (Masaki et al., Agric Biol Chem 42:1443-5, 1978), and Myxobacteria strain AL-1 (Wingard et al., JBact 112:940-9, 1979). This protease is frequently used alone or in combination with other proteases for a variety of applications including in-solution or in-gel protein digestion, phosphopeptide enrichment, protein mapping, peptide mass fingerprinting, mass spectrometry-based spectral matching, and proteomics.

[0043] The present disclosure provides mutant variants of Lys-C proteases that exhibit enhanced autolysis resistance. The disclosed Lys-C proteases can be obtained or derived from any biological source, including bacteria and / or artificial expression or synthesis systems. In some embodiments, the Lys-C protease is obtained from or derived from Achromobacterlyticus. In some embodiments, the Lys C-protease is obtained from or derived from Lysobacter spp. In some embodiments, Lysobacter spp. is selected from Lysobacter enzymogenes, Lysobacter antibiotus, Lysobacter sp. Root96 and Lysobacter maris. In some embodiments, the Lys-C protease is obtained from or derived from Myxobacteria strain AL-1. In some embodiments, the Lys-C protease is obtained from or derived from Shewanella spp. In some embodiments, the Lys-C protease is obtained or derived from Aquimonas. In some embodiments, the Lys-C protease is obtained or derived from Pseudofulvimonas. In some embodiments, the Lys-C protease is obtained or derived from Tahibacter. In some embodiments, the Lys-C protease is obtained or derived from Thalassocella.

[0044] The wild-type amino acid sequence of Lys-C of Achromobacter lyticus is provided in SEQ ID NO:1 below, with bold letters distinguishing lysine (Lys; K) residues at amino acid positions 2, 39, 52, 54, 62, 104, 173, 178, 183, 205, 235, 254, 311, 360 and 408, which serve as the natural substrate for the proteolytic active site of the enzyme.

[0045]

[0046] Lys-C protease includes a protease domain that performs its enzymatic function. The amino acid sequence of the wild-type Lys-C protease domain is provided in SEQ ID NO:2 below, with bold letters distinguishing the Lys residues at amino acid positions 30, 49, 106, 155 and 203, which serve as the natural substrate for the proteolytic active site of the enzyme.

[0047]

[0048]

[0049] In some embodiments, the wild-type Lys-C enzyme is modified to incorporate one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more) conservative amino acid substitutions. In some embodiments, the wild-type Lys-C enzyme is modified to incorporate one conservative amino acid substitution. In some embodiments, the wild-type Lys-C enzyme is modified to incorporate two conservative amino acid substitutions. In some embodiments, the wild-type Lys-C enzyme is modified to incorporate three conservative amino acid substitutions. In some embodiments, the wild-type Lys-C enzyme is modified to incorporate four conservative amino acid substitutions. In some embodiments, the wild-type Lys-C enzyme is modified to incorporate five conservative amino acid substitutions. In some embodiments, the wild-type Lys-C enzyme is modified to incorporate six conservative amino acid substitutions. In some embodiments, one or more conservative amino acid substitutions are lysine to arginine (Arg) substitutions. In some embodiments, one or more conservative substitutions include a Lys to Arg substitution at one or more amino acid residues selected from residues 2, 39, 52, 54, 62, 104, 173, 178, 183, 205, 235, 254, 311, 360, and 408 of SEQ ID NO: 1, or a combination thereof, or at one or more amino acid residues selected from residues 30, 49, 106, 155, and 203 of SEQ ID NO: 2, or a combination thereof. In some embodiments, one or more conservative substitutions include a Lys to Arg substitution at amino acid residue 2 of SEQ ID NO: 1. In some embodiments, one or more conservative substitutions include a Lys to Arg substitution at amino acid residue 39 of SEQ ID NO: 1. In some embodiments, one or more conservative substitutions include a Lys to Arg substitution at amino acid residue 52 of SEQ ID NO: 1. In some embodiments, one or more conservative substitutions include a Lys to Arg substitution at amino acid residue 54 of SEQ ID NO: 1. In some embodiments, one or more conservative substitutions include a Lys to Arg substitution at amino acid residue 62 of SEQ ID NO: 1. In some embodiments, one or more conservative substitutions include a Lys to Arg substitution at amino acid residue 104 of SEQ ID NO: 1. In some embodiments, one or more conservative substitutions include a Lys to Arg substitution at amino acid residue 173 of SEQ ID NO: 1. In some embodiments, one or more conservative substitutions include a Lys to Arg substitution at amino acid residue 178 of SEQ ID NO: 1. In some embodiments, one or more conservative substitutions include a Lys to Arg substitution at amino acid residue 183 of SEQ ID NO: 1.In some embodiments, one or more conservative substitutions include a Lys to Arg substitution at amino acid residue 205 of SEQ ID NO: 1. In some embodiments, one or more conservative substitutions include a Lys to Arg substitution at amino acid residue 235 of SEQ ID NO: 1 or amino acid residue 30 of SEQ ID NO: 2. Alternatively or in addition, one or more conservative substitutions include a Lys to Arg substitution at amino acid residue 254 of SEQ ID NO: 1 or amino acid residue 49 of SEQ ID NO: 2. Alternatively or in addition, one or more conservative substitutions include a Lys to Arg substitution at amino acid residue 311 of SEQ ID NO: 1 or amino acid residue 106 of SEQ ID NO: 2. Alternatively or in addition, one or more conservative substitutions include a Lys to Arg substitution at amino acid residue 360 ​​of SEQ ID NO: 1 or amino acid residue 155 of SEQ ID NO: 2. Alternatively or additionally, the one or more conservative substitutions include a Lys to Arg substitution at amino acid residue 408 of SEQ ID NO:1 or amino acid residue 203 of SEQ ID NO:2. In some embodiments, the wild-type Lys-C protease is modified with conservative substitutions of Lys to Arg at amino acid residues 2, 39, 52, 54, 62, 104, 173, 178, 183, 205, 235, 254, 311, 360 and 408 of SEQ ID NO: 1 to generate a mutant Lys-C protease having the amino acid sequence of SEQ ID NO: 3, with the Arg (substituted from Lys) residues at amino acid positions 2, 39, 52, 54, 62, 104, 173, 178, 183, 205, 235, 254, 311, 360 and 408 being distinguished by bold letters, which individually and / or collectively confer enhanced resistance to autolysis to the mutant Lys-C protease.

[0050]

[0051] In some embodiments, the wild-type Lys-C protease domain is modified with conservative substitutions of Lys to Arg at amino acid residues 30, 49, 106, 155, and 203 of SEQ ID NO: 2 to generate a mutant Lys-C protease domain having an amino acid sequence of SEQ ID NO: 4, with bold letters distinguishing the Arg (substituted from Lys) residues at amino acid positions 30, 49, 106, 155, and 203, which alone and / or together confer enhanced autolysis resistance to the mutant Lys-C protease domain. The Arg residue is not part of the active site of Lys-C. Therefore, it is expected that these mutations do not change the activity of Lys-C.

[0052]

[0053] In some embodiments, the Lys-C protease can be modified with one or more of the above-described conservative amino acid residues so that the protease exhibits, for example, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% increased resistance to autolysis compared to the autolysis resistance of the Lys-C protease in the absence of the one or more conservative substitutions.

[0054] Asp-N

[0055] Endoprotease Asp-N (about 25 kDa; also known as flavastacin) is a zinc metalloprotease that specifically cleaves peptide bonds on the N-terminal side of aspartic acid (Asp; D) and cysteine ​​(Cys; C) residues. Asp-N exhibits optimal protease activity in the pH range of 4.0-9.0 and is highly resistant to strong denaturing conditions (e.g., high concentrations of urea). Asp-N is typically derived from mutant Pseudomonas fragi strains (Ingrosso et al., Biochem Biophys Res Commun. 162: 1528-34, 1989) and Stenotrophomonas maltophilia. This protease is often used alone or in combination with other proteases for a variety of applications, including protein digestion in solution or in gel, phosphopeptide enrichment, protein mapping, peptide mass fingerprinting, mass spectrometry-based spectral matching, and proteomics.

[0056] The present disclosure provides mutant variants of Asp-N proteases that exhibit enhanced autolysis resistance. The disclosed Asp-N proteases can be obtained from or derived from any biological source, including bacteria and / or artificial expression or synthesis systems. In some embodiments, the Asp-N protease is obtained from or derived from Stenotrophomonas maltophilia.

[0057] The wild-type amino acid sequence of Asp-N from Stenotrophomonas maltophilia is provided below in SEQ ID NO:5, with bold letters distinguishing aspartic acid (Asp; D) residues at amino acid positions 2, 14, 46, 67, 71 and 130, which serve as the natural substrate for the proteolytic active site of the enzyme.

[0058]

[0059] In some embodiments, the wild-type Asp-N enzyme is modified to incorporate one or more (e.g., 1, 2, 3, 4, 5, 6 or more) conservative amino acid substitutions. In some embodiments, the wild-type Asp-N enzyme is modified to incorporate one conservative amino acid substitution. In some embodiments, the wild-type Asp-N enzyme is modified to incorporate two conservative amino acid substitutions. In some embodiments, the wild-type Asp-N enzyme is modified to incorporate three conservative amino acid substitutions. In some embodiments, the wild-type Asp-N enzyme is modified to incorporate four conservative amino acid substitutions. In some embodiments, the wild-type Asp-N enzyme is modified to incorporate five conservative amino acid substitutions. In some embodiments, the wild-type Asp-N enzyme is modified to incorporate six conservative amino acid substitutions. In some embodiments, one or more conservative amino acid substitutions are substitutions of Asp to glutamic acid (Glu; E). In some embodiments, one or more conservative substitutions include substitutions of Asp to Glu at one or more amino acid residues selected from residues 2, 14, 46, 67, 71 and 130 of SEQ ID NO:5, or a combination thereof. In some embodiments, one or more conservative substitutions include a substitution of Asp to Glu at amino acid residue 2 of SEQ ID NO:5. Alternatively or in addition, one or more conservative substitutions include a substitution of Asp to Glu at amino acid residue 14 of SEQ ID NO:5. Alternatively or in addition, one or more conservative substitutions include a substitution of Asp to Glu at amino acid residue 46 of SEQ ID NO:5. Alternatively or in addition, one or more conservative substitutions include a substitution of Asp to Glu at amino acid residue 67 of SEQ ID NO:5. Alternatively or in addition, one or more conservative substitutions include a substitution of Asp to Glu at amino acid residue 71 of SEQ ID NO:5. Alternatively or in addition, one or more conservative substitutions include a substitution of Asp to Glu at amino acid residue 130 of SEQ ID NO:5. Alternatively or in addition, the wild-type Asp-N protease is modified with conservative substitutions from Asp to Glu at amino acid residues 2, 14, 46, 67, 71 and 130 of SEQ ID NO:5 to produce a mutant Asp-N protease having the amino acid sequence of SEQ ID NO:6, with the Glu (substituted from Asp) residues at amino acid positions 2, 14, 46, 67, 71 and 130 being distinguished by bold letters, which individually and / or collectively confer enhanced resistance to autolysis to the mutant Asp-N protease.

[0060]

[0061] In some embodiments, the wild-type Asp-N protease is not modified by one or more (e.g., 1, 2, or more) conservative substitutions from Asp to Glu at the active site of the protease (including amino acid positions 67 and 71 of SEQ ID NO:5). Thus, in some embodiments, the wild-type Asp-N protease is modified with conservative substitutions from Asp to Glu at amino acid residues 2, 14, 46, and 130 of SEQ ID NO:5 to generate a mutant Asp-N protease having the amino acid sequence of SEQ ID NO:7, with bold letters distinguishing the Glu (substituted from Asp) residues at amino acid positions 2, 14, 46, and 130, which individually and / or collectively confer enhanced autolysis resistance to the mutant Asp-N protease. Bold and underlined letters distinguish the wild-type Asp residues (amino acid positions 67 and 71) within the Asp-N active site.

[0062]

[0063] In some embodiments, the Asp-N protease can be modified with one or more of the above-described conservative amino acid residues such that the protease exhibits, for example, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% increased resistance to autolysis compared to the autolysis resistance of the Asp-N protease in the absence of the one or more conservative substitutions.

[0064] Trypsin

[0065] Trypsin (about 24kDa) is a widely used serine protease for peptide mapping analysis and proteomics research based on LC-MS. The enzyme performs its proteolytic function by cleaving the peptide chain at the carboxyl side of Lys or Arg residues. The enzyme is synthesized as an inactive precursor trypsinogen in the pancreatic cells of vertebrates and is subsequently converted into an active form by cleaving the propeptide. The activation process can also be carried out autocatalytically at physiological pH. Trypsin is mainly used for peptide trypsin cleavage into small slices for sequencing, separation of adherent cells from coated cell culture dishes, cleavage of fusion proteins, activation of zymogens (e.g., from trypsinogen to trypsin) and for the recombinant production of peptide hormones. Trypsin is also a component of some pharmaceutical preparations (e.g., ointments, lozenges, and inhalation aerosols).

[0066] Some efforts have been made to improve its analytical capabilities. Trypsin was originally derived from the purification of animal source materials for analytical work. Porcine trypsin has become preferred for analytical applications, although cattle and human variants are also commercially available. Recombinantly expressed and affinity purified trypsin is now widely commercially available. In addition, the practice of chemically alkylating (e.g., methylating) trypsin on the Lys residue of trypsin has become a widely accepted practice to reduce autolysis during sample preparation. However, the trypsin sequence also contains Arg residues, which are the same as Lys, natural substrates for trypsin proteolysis. These Arg residues are subject to the action of the trypsin active site, and even to the action of the amino-alkylated derivative form of the enzyme.

[0067] The present disclosure provides mutant variants of trypsin that exhibit enhanced resistance to autolysis. The disclosed trypsin can be obtained from or derived from any biological source, including bacteria and fungi (e.g., Streptomyces, Escherichia coli, Pichia pastoris, H. Polymorpha, Saccharomyces cerevisiae, and S. pombae), mammalian sources (e.g., cattle, pigs, birds, mice, rodents, and humans), plant sources, and / or artificial expression or synthetic systems. In some embodiments, trypsin is bovine trypsin. In some embodiments, trypsin is porcine trypsin. In some embodiments, trypsin is avian trypsin. In some embodiments, trypsin is rat trypsin. In some embodiments, trypsin is rodent trypsin. In some embodiments, trypsin is human trypsin.

[0068] The wild-type amino acid sequence of porcine trypsin is provided below in SEQ ID NO:8, wherein bold letters distinguish Arg residues at amino acid positions 45, 49, 99 and 107, which serve as natural substrates for the proteolytic active site of the enzyme, and underlined letters correspond to Lys residues at amino acid positions 89, 125, 139, 149, 170, 200, 202 and 208, which can be alkylated (e.g., methylated) to further enhance resistance to autolysis.

[0069]

[0070] In some embodiments, wild-type trypsin is modified to incorporate one or more (e.g., 1, 2, 3, 4 or more) conservative amino acid substitutions. In some embodiments, wild-type trypsin is modified to incorporate one conservative amino acid substitution. In some embodiments, wild-type trypsin is modified to incorporate two conservative amino acid substitutions. In some embodiments, wild-type trypsin is modified to incorporate three conservative amino acid substitutions. In some embodiments, wild-type trypsin is modified to incorporate four conservative amino acid substitutions. In some embodiments, wild-type trypsin is modified to incorporate five conservative amino acid substitutions. In some embodiments, wild-type trypsin is modified to incorporate six conservative amino acid substitutions. In some embodiments, one or more conservative amino acid substitutions are substitutions of Arg to Lys. In some embodiments, one or more conservative substitutions include substitutions of Arg to Lys at one or more amino acid residues selected from residues 45, 49, 99 and 107 of SEQ ID NO:8, or combinations thereof. In some embodiments, one or more conservative substitutions include substitutions of Arg to Lys at amino acid residue 45 of SEQ ID NO:8. Alternatively or additionally, the one or more conservative substitutions include an Arg to Lys substitution at amino acid residue 49 of SEQ ID NO: 8. Alternatively or additionally, the one or more conservative substitutions include an Arg to Lys substitution at amino acid residue 99 of SEQ ID NO: 8. Alternatively or additionally, the one or more conservative substitutions include an Arg to Lys substitution at amino acid residue 107 of SEQ ID NO: 8. Alternatively or in addition, wild-type trypsin is modified with conservative substitutions of Arg to Lys at amino acid residues 45, 49, 99 and 107 of SEQ ID NO: 8 to produce a mutant trypsin having the amino acid sequence of SEQ ID NO: 9, wherein bold letters distinguish Lys (substituted from Arg) residues at amino acid positions 45, 49, 99 and 107, which confer enhanced autolysis resistance to the mutant trypsin alone and / or together, and underlined letters correspond to Lys residues at amino acid positions 89, 125, 139, 149, 170, 200, 202 and 208, which can be alkylated (e.g., methylated) to further enhance autolysis resistance. The Arg residue is not part of the active site of trypsin. Therefore, it is expected that these mutations do not change the proteolytic activity of the enzyme.

[0071]

[0072] In some embodiments, wild-type trypsin can be modified with one or more of the above-described conservative amino acid residues such that the protease exhibits, for example, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% increased resistance to autolysis compared to the autolysis resistance of trypsin in the absence of the one or more conservative substitutions.

[0073] Chemical modification of recombinant proteases

[0074] The autolysis resistance of proteases (e.g., Lys-C, Asp-N, and trypsin) genetically engineered to incorporate one or more (e.g., 1, 2, 3, 4, 5, 6, or more) conservative amino acid substitutions can be further enhanced by adding chemical modifications to one or more (e.g., 1, 2, 3, 4, 5, 6, or more) amino acid residues of the natural substrate that is the active site of the enzyme, or to one or more amino acid residues that have been mutated via conservative amino acid substitutions to enhance autolysis resistance. In some embodiments, the chemical modification is selected from an alkyl moiety, an acetyl moiety, an amide moiety, an ester moiety, an imine moiety, an amidino moiety, a guanidino moiety, or a thioether moiety. For example, a well-known method for improving the autolysis resistance of trypsin is to add an alkyl group (e.g., a monoalkyl or dialkyl group) at a Lys residue (Rice et al., Biochim Biophys Acta. 492: 316-21, 1977; and Means et al., Biochem. 7: 2192-2210, 1968).

[0075] Thus, the present disclosure provides compositions and methods for alkylating the recombinant proteases of the present disclosure (e.g., Lys-C, Asp-N, and trypsin) to further improve the autolysis resistance of the enzymes. The alkyl group can be attached to the amine group of the amino acid residue (e.g., Lys) of the enzyme. In some embodiments, the alkyl group can be a primary or branched C 1-12 In some embodiments, the recombinant protease of the present disclosure is wherein the alkyl group is a primary or branched C 1-4Those of alkyl groups. The alkylation of proteases is usually carried out by reductive alkylation. The degree of alkylation of amino acid residues will depend on the reaction conditions of the reductive alkylation process. For example, if the reaction cycle is repeated multiple times and / or a higher reagent: enzyme ratio is used, complete alkylation, i.e., alkylation of all target residues, will be achieved. In some embodiments, the recombinant protease of the present invention can be completely dialkylated at all its target amino acid residues. In some embodiments, the recombinant protease of the present invention can be partially alkylated at one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 or more) target amino acid residues. In some embodiments, the alkyl moiety is selected from a methyl moiety, a dimethyl moiety, an octanal moiety, and a cyclodextrin monoaldehyde moiety. An exemplary, non-limiting method for the reductive methylation of trypsin is described in Example 2.

[0076] Expression and purification of recombinant proteases

[0077] Expression of recombinant proteases

[0078] Recombinant expression systems can be used to produce recombinant proteases disclosed herein (e.g., Lys-C, Asp-N and trypsin). For example, polynucleotides (e.g., DNA or RNA) encoding mutant proteases of the present disclosure can be incorporated into a recombinant expression vector that can support and promote the expression of proteases in host cells.

[0079] In one non-limiting example, disclosed herein is a method for expressing an engineered protease of the present disclosure using a recombinant expression system, the method comprising: (1) transforming a host cell with a recombinant nucleic acid comprising a sequence encoding a mutant protease from a bacterial, fungal, plant or mammalian source; and (2) culturing the host cell under conditions and for a time sufficient to allow stable expression of the protease; and (3) isolating the expression product from the culture medium.

[0080] The exemplary methods that can be used to realize the expression of one or more recombinant proteases of the present disclosure in host cells are described in further detail below. A platform that can be used to obtain the effective intracellular concentration of one or more proteases described herein in host cells is via the stable expression of genes encoding these enzymes (for example, by being integrated into the nuclear or mitochondrial genome of the host cell). These genes are polynucleotides encoding the primary amino acid sequence of the corresponding protein. In order to introduce these exogenous genes into the host cells, these genes can be integrated into the vector. By a variety of methods, including conversion, transfection, direct uptake, projectile bombardment and by encapsulating the vector in liposomes, the vector is introduced into the cell. The example of the suitable method of transfection or transformed cells is calcium phosphate precipitation, electroporation, microinjection, infection, lipofection and direct uptake. Such methods are described in more detail in, for example, Green et al., Molecular Cloning: A Laboratory Manual, Fourth Edition (Cold Spring Harbor University Press, New York (2014)); and Ausubel et al., Current Protocols in Molecular Biology (John Wiley & Sons, New York (2015)), the disclosures of each of which are incorporated herein by reference.

[0081] Genes encoding therapeutic proteins of the present disclosure may also be introduced into host cells by targeting vectors containing genes encoding such agents to cell membrane phospholipids.

[0082] For gene expression, RNA polymerase is important to the identification and combination of the polynucleotide of one or more proteases of the present disclosure. Therefore, it is possible to include in the polynucleotide a sequence element that raises RNA polymerase and promotes the transcription factor of transcription complex to assemble at the transcription start site and shows high affinity. Such sequence element includes, for example, a promoter, whose sequence can be recognized and combined by specific transcription initiation factor and final RNA polymerase, and it is operably connected to a protease encoding sequence (for example, upstream thereof). The general example of the promoter type applicable to the disclosed composition and method includes constitutive promoter, spatiotemporal promoter, inducible promoter and synthetic promoter. Examples of suitable promoters for directing transcription of nucleic acids encoding one or more proteases of the present disclosure, particularly in bacterial hosts, are the promoter of the E. coli lac operon, the Streptomyces coelicolor agarase gene dagA promoter, the Bacillus licheniformis α-amylase gene (amyL) promoter, the Bacillus stearothermophilus maltogenic amylase gene (amyM) promoter, the Bacillus Amyloliquefaciens α-amylase (amyQ) promoter, the Bacillus subtilis xylA and xylB gene promoters, and the like. For transcription in a fungal host, examples of useful promoters are those derived from genes encoding A. oryzae TAKA amylase, Rhizomucor miehei aspartic proteinase, A. niger neutral α-amylase, A. niger acid-stable α-amylase, A. niger glucoamylase, Rhizomucor miehei lipase, A. oryzae alkaline proteinase, A. oryzae triose phosphate isomerase, or A. nidulans acetamidase. Non-limiting examples of mammalian promoters include a cytomegalovirus (CMV) promoter, a CAG promoter, an elongation factor 1 alpha (EF1α) promoter, a eukaryotic elongation factor 2 (EEF2) promoter, a glyceraldehyde 3-phosphate dehydrogenase (GAPDH) promoter, a phosphoglycerate kinase (PGK) promoter, an actin promoter (e.g., CBA), a CK8 promoter, a TBG promoter, an H1 promoter, a 7SK promoter, and an ubiquitin promoter.For transcription in a plant host, non-exemplary of useful promoters are CaMV35S cauliflower mosaic virus promoter, actin promoter, ubiquitin (e.g., GMUBI3, GMUBI7, RUBQ2, RUBI3, ZMUBI1, UBB1, PUVBI1, and UBI7) promoter, tubulin promoter, eukaryotic initiation factor (EIF) promoter, ascorbate peroxidase (APX) promoter, phosphogluconate dehydrogenase (PGD1) promoter, R1G1 domain-containing protein B (R1G1B) promoter, dehydration element binding (DREB1a) promoter, salt tolerance zinc finger (STZ / ZAT10) promoter, wax production 1 (WXP1) promoter, prolamin promoter, glutenin promoter, cell wall relaxin promoter, ACC-oxidase promoter, E8 promoter, polygalacturonase (PG) promoter, dioscorin promoter, pDJ3S promoter, potato class I potato glycoprotein promoter, GBSS granule-bound starch synthase promoter, sweet potato root tuber specific protein promoter, β-amylase, RA8 promoter, A9 promoter, TA29 promoter, OSNCED3 promoter, WSI18 promoter, R29A promoter and OSPR10 promoter. Various viral promoters and additional regulatory elements (e.g., enhancers, terminators, etc.) can also be used to direct transcription of nucleic acids encoding one or more proteases of the present disclosure, including, for example, bacteriophage T7, T3 and SP6 promoters and / or terminator elements, etc.

[0083] Once the polynucleotide encoding one or more recombinant proteases of the present disclosure has been internalized and / or incorporated into the nuclear DNA of the host cell outside the chromosome by the host cell, the transcription of the polynucleotide can be induced by methods known in the art. For example, expression can be induced by exposing the host cell to an external chemical reagent, such as a reagent that regulates the binding of transcription factors and / or RNA polymerases to promoters to regulate gene expression. Chemical reagents can be used to promote the binding of RNA polymerases and / or transcription factors to promoters, such as by removing the repressor protein that has been bound to the promoter. Alternatively, chemical reagents can be used to enhance the affinity of promoters to RNA polymerases and / or transcription factors, so that the transcription rate of genes located downstream of the promoter is increased in the presence of chemical reagents. Examples of chemical reagents that strengthen polynucleotide transcription by the above-mentioned mechanism are tetracycline and doxycycline. These reagents are commercially available (Life Technologies, Carlsbad, CA), and can be applied to host cells according to established protocols to promote gene expression.

[0084]

[00136] Other DNA sequence elements that may be included in polynucleotides for use in the compositions and methods described herein are enhancer sequences.

[0085] Enhancers represent another class of regulatory elements that induce conformational changes in polynucleotides containing genes of interest, causing DNA to adopt a three-dimensional orientation that is conducive to the binding of transcription factors and RNA polymerases at the transcription start site. Therefore, polynucleotides for compositions and methods described herein include those encoding one or more proteases disclosed herein, and also include enhancer sequences. Many enhancer sequences from bacteria, avian, and mammalian genes are now known. Non-limiting examples are enhancers from genes encoding mammalian globulins, elastase, albumin, α-fetoprotein, and insulin. Enhancers for compositions and methods described herein also include those derived from genetic material of viruses capable of infecting eukaryotic cells. Examples are SV40 enhancers (bp 100-270) in the late replication start point, cytomegalovirus early promoter enhancers, polyoma enhancers in the late replication start point, and adenovirus enhancers. Other enhancer sequences that induce transcriptional activation of eukaryotic genes are disclosed in Yaniv et al., Nature 297:17 (1982).

[0086] Expression vector

[0087] A variety of vectors have been developed for delivering polynucleotides encoding exogenous proteins to host cells. Expression vectors for the compositions and methods described herein may contain one or more polynucleotides encoding one or more proteases of the present disclosure, and may also include, for example, one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) nucleic acid elements for regulating expression of these agents and / or integration of such polynucleotides into the host cell genome.

[0088] In some embodiments, the vector can be an autonomously replicating vector, that is, a vector that exists as an extrachromosomal entity, and its replication is independent of chromosomal replication, such as a plasmid, a phage, an extrachromosomal element, a minichromosome, or an artificial chromosome. Alternatively, the vector can be such a vector: when it is introduced into a host cell, it is integrated into the host cell genome and replicated together with the chromosome into which it is integrated. Certain vectors that can be used to express one or more engineered proteases described herein include plasmids containing regulatory sequences such as promoters and optional enhancer regions that direct gene transcription. Other useful vectors for expressing one or more proteases disclosed herein contain polynucleotide sequences that enhance the translation rate of these genes or improve the stability or nuclear export of mRNA produced by gene transcription. These sequence elements include, for example, 5' and 3' untranslated regions, internal ribosome entry sites (IRES), and polyadenylation signal sites to guide the efficient transcription of genes carried on the expression vector. Expression vectors suitable for compositions and methods described herein may also contain polynucleotides encoding markers that are used to select cells containing such vectors. Examples of suitable markers are genes encoding resistance to antibiotics such as ampicillin, chloramphenicol, kanamycin and nourseothricin, among others.

[0089] In some embodiments, the expression vector of the present disclosure further comprises a polynucleotide encoding a protein tag, such as a His tag (e.g., 6x-His (SEQ ID NO: 10)), a maltose binding protein tag, a SNAP tag, a FLAG tag, a halotag, a fluorescent protein tag, and the like.

[0090] Viral vectors

[0091] Viral genomes provide a rich source of vectors that can be used to efficiently deliver exogenous genes to host cells. Viral genomes are particularly useful vectors for gene delivery because the polynucleotides contained in such genomes are usually incorporated into the nuclear genome of host cells by universal transduction and localized transduction. These processes occur as part of the natural viral replication cycle and do not require the addition of proteins or reagents to induce gene integration. Examples of viral vectors are retroviruses (e.g., retroviridae family viral vectors, adenoviruses (e.g., Ad5, Ad26, Ad34, Ad35, and Ad48), parvoviruses (e.g., adeno-associated virus), coronaviruses, negative-strand RNA viruses such as orthomyxoviruses (e.g., influenza virus), rhabdoviruses (e.g., rabies and vesicular stomatitis virus), paramyxoviruses (e.g., measles and Sendai virus), positive-strand RNA viruses such as picornaviruses and alphaviruses, and double-stranded DNA viruses including adenoviruses, herpes viruses (e.g., herpes simplex virus type 1 and type 2, Epstein-Barr virus, and cytomegalovirus), and pox viruses (e.g., vaccinia virus, modified vaccinia Ankara virus (MVA), fowlpox virus, and canarypox virus). Viruses). Other viruses include, for example, Norwalk virus, togavirus, flavivirus, reovirus, papovavirus, hepadnavirus, human papillomavirus, human foamy virus, and hepatitis virus. Examples of retroviruses include, but are not limited to, avian leukosis-sarcoma, avian C-type virus, mammalian C-type, B-type virus, D-type virus, oncorretrovirus, HTLV-BLV group, lentivirus, alpha retrovirus, gamma retrovirus, foamy virus. Other examples are mouse mammary tumor virus, mouse sarcoma virus, mouse leukemia virus, bovine leukemia virus, feline leukemia, feline sarcoma virus, avian leukemia virus, human T-cell leukemia virus, baboon endogenous virus, gibbon ape leukemia virus, Mason Pfizer monkey virus, simian immunodeficiency virus, simian sarcoma virus, Rous sarcoma virus, and lentivirus.Plant viruses may also be suitable for use with the compositions and methods disclosed herein, including but not limited to double-stranded DNA viruses such as cauliflower mosaic virus and baculovirus, single-stranded DNA viruses such as Geminiviridae, and RNA viruses such as Reoviridae (e.g., phytoreovirus, fijivirus, and oryzavirus), partiviridae (e.g., alphalatent virus and betalatent virus), Rhabdoviridae (e.g., intracellular rice yellow dwarf cannonball virus and intranuclear rice yellow dwarf cannonball virus), Bunyaviridae (e.g., tomato spotted wilt virus), teniviridae, Associaviridae (e.g., tomato bushy stunt virus), dianthusvirus, The vectors include, for example, chlorosis virus, maize chlorotic mottle virus, maize rhadovirus, necrovirus, southern bean mosaic virus, turnip yellow mosaic virus, pea ear mosaic virus, rubus virus, brome mosaic virus (e.g., cucumovirus, brome mosaic virus, ilavirus, alfalfa mosaic virus), cowpea mosaic virus (e.g., nematode-transmitted polyhedron virus), tobacco mosaic virus, tobacco rattle virus, barley virus, fungus-transmitted baculovirus, potex virus, morphovirus, trichovirus, cartalvirus, potyviridae, and closterovirus. Other examples of vectors are described, for example, in US 5,801,030A, the disclosure of which is incorporated herein by reference in its entirety.

[0092] Methods for delivering recombinant nucleic acids to host cells

[0093] Techniques that can be used to introduce polynucleotides (e.g., polynucleotides encoding one or more recombinant proteases disclosed herein) into host cells are well known in the art. For example, electroporation can be used to permeabilize host cells by applying an electrostatic potential to the cells of interest. Host cells subjected to an external electric field in this manner subsequently tend to take up exogenous nucleic acids. Similar techniques, Nucleofection TM , using an applied electric field to stimulate the uptake of exogenous polynucleotides into the nucleus of eukaryotic cells.

[0094] Another technique for target cell transfection is the extrusion-perforation method. This technique induces rapid mechanical deformation of cells to stimulate the uptake of exogenous DNA through membrane pores that are shaped in response to the applied stress. The advantage of this technique is that no carrier is required to deliver nucleic acid to cells (such as target cells).

[0095] Liposome infection represents another technology that can be used for transfecting target cells. The method involves loading nucleic acid into liposomes, which generally provide cationic functional groups, such as quaternary ammonium or protonated amines, to the outside of the liposomes. This results in the uptake of exogenous nucleic acids, such as by direct fusion of liposomes with cell membranes or by endocytosis of complexes. A similar technique for stimulating exogenous nucleic acid uptake using ionic interactions with cell membranes is to contact cells with cationic polymer-nucleic acid complexes. Associated with polynucleotides to impart an exemplary cationic molecule that is conducive to interacting with cell membranes is activated dendrimers, polyethyleneimine, and diethylaminoethyl (DEAE)-dextran. Magnetic beads are another tool that can be used for transfecting target cells in a gentle and effective manner, because the method step utilizes an applied magnetic field to guide the uptake of nucleic acids.

[0096] Another useful tool for inducing uptake of exogenous nucleic acids by target cells is laser transfection, also known as optical transfection, a technique that involves exposing cells to electromagnetic radiation of a specific wavelength to gently permeabilize the cells and allow polynucleotides to penetrate the cell membrane. The biological activity of this technique is similar to, and in some cases has been found to be superior to, electroporation.

[0097] Puncture infection is another technique that can be used to deliver genetic material to target cells. It relies on the use of nanomaterials, such as carbon nanofibers, carbon nanotubes, and nanowires. Needle-shaped nanostructures are synthesized perpendicular to the substrate surface. DNA containing genes for intracellular delivery is attached to the nanostructure surface. A chip with an array of these needles is then pressed against cells or tissues. Cells pierced by the nanostructures can express the delivered genes.

[0098] Magnetofection can also be used to deliver nucleic acids to target cells. The magnetofection principle is to bind nucleic acids to cationic magnetic nanoparticles. The magnetic nanoparticles are made of iron oxide, which is completely biodegradable and coated with specific cationic proprietary molecules that vary depending on the application. Their binding to the nucleic acid carrier is achieved by salt-induced colloidal aggregation and electrostatic interactions. The magnetic particles are then concentrated on the target cells by the influence of an external magnetic field generated by a magnet.

[0099] Another useful tool for inducing uptake of exogenous nucleic acids by target cells is sonoporation, a technique involving the use of sound (usually ultrasonic frequencies) to alter the permeability of the cellular plasma membrane to permeabilize the cell and allow the polynucleotide to penetrate the cell membrane.

[0100] Microvesicles represent another potential carrier that can be used to modify the target cell genome according to the methods described herein. For example, microvesicles induced by co-overexpression of glycoprotein VSV-G and, for example, genome modification proteins (such as nucleases) can be used to effectively deliver proteins into cells, which then catalyze site-specific cleavage of endogenous polynucleotide sequences to prepare the cell genome for covalent incorporation of polynucleotides of interest such as genes or regulatory sequences.

[0101] Protease purification

[0102] After expressing the disclosed protease in a cell-based or cell-free manner using the methods disclosed herein, the protease is purified and separated for use. A variety of well-known protein purification methods can be used in combination with the disclosed method. In a cell-based expression system, purification usually starts with the preparation of a crude extract containing a complex mixture of all proteins from the cytoplasm and various other macromolecules, cofactors and nutrients. In some embodiments, a crude extract is prepared using a chemical method, an enzymatic method, an ultrasonic treatment or a French press. Subsequently, debris can be removed from the crude extract by centrifugation, and the supernatant containing the expressed protease is recovered. Various well-known methods can then be used to separate the recombinant protein from the supernatant, including but not limited to chromatographic methods (e.g., affinity chromatography, HPLC, SEC and IEC), protein precipitation, and cation exchange and gel filtration. The confirmation of protein purity can be carried out by well-known methods, including, for example, HPLC, MS, SDS-PAGE, ELISA, Bradford assay, UV-visible spectroscopy, activity assay, dynamic light scattering, microfluidic diffusion measurement technology, sedimentation velocity method and immunoblotting.

[0103] Host cells

[0104] Host cells suitable for use with the disclosed compositions and methods may be eukaryotic host cells or prokaryotic host cells well known in the art and routinely used for recombinant protein expression. Host cells of the present disclosure comprising polynucleotides of the present disclosure or expression vectors as defined herein are advantageously used as host cells for recombinant production of proteases of the present disclosure. In some embodiments, cells of the present disclosure may be cells of higher organisms such as mammals or insects, but may also be microbial cells such as bacteria or fungi (including yeast, such as budding yeast and brewing yeast) cells or plant cells.

[0105] In some embodiments, the host cell is a mammalian cell. The mammalian cell can be, but is not limited to, a Chinese hamster ovary (CHO) cell, a human embryonic kidney 293 (HEK293) cell, and a HEK293T cell. In some embodiments, the host cell is an insect cell (e.g., Spodopterafrugiperda, such as SF9 cell).

[0106] Alternatively, the host cell is a bacterial cell. Examples of suitable bacterial cells are Gram-positive bacteria, such as Bacillus subtilis, Bacillus licheniformis, Bacillus lentus, Bacillus brevis, Bacillus stearothermophilus, Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus coagulans, Bacillus circulans, Bacillus lautus, Bacillus megaterium, Bacillus thuringiensis, Streptomyces lividans, or Streptomyces murinus, or Gram-negative bacteria such as E. coli. Transformation of bacteria can be achieved, for example, by protoplast transformation or by using competent cells in a manner known per se.Example 1 provides an exemplary method for recombinant expression of a protease of the present disclosure in E. coli.

[0107] Alternatively, the host cell is a fungal cell. In some embodiments, the fungal cell is a yeast cell. In some embodiments, the yeast is selected from Pichia pastoris, Hansenula polymorpha, Saccharomyces cerevisiae, Schizosaccharomyces pombae.

[0108] Cell-free expression system

[0109] Alternatively, the recombinant proteases of the present disclosure can be expressed in vitro using a cell-free expression system that facilitates the production of recombinant proteins without the use of living cells. Typically, a cell-free expression system includes a solution containing components necessary to direct protein synthesis, such as polynucleotides encoding proteins, ribosomes, tRNA, enzymes, cofactors, amino acids, etc. Non-limiting examples of cell-free expression systems include Cell-free Escherichia coli protein synthesis system ( Cell-free E.coli Protein Synthesis System) and In vitro protein synthesis kit ( In Vitro Protein Synthesis kit) etc.

[0110] Alternatively or in addition, the recombinant protease of the present disclosure can be synthesized, for example, using solid phase peptide synthesis. Solid phase peptide synthesis is a method for chemically synthesizing peptides on a solid support. In solid phase peptide synthesis, an amino acid or peptide is usually attached to a solid support via the C-terminus. New amino acids are added to the attached amino acids or peptides by a coupling reaction. Due to the possibility of unexpected reactions, protecting groups are usually used. To date, solid phase peptide synthesis has become a standard operation for chemical peptide synthesis.

[0111] Assays for assessing autolysis and target proteolysis

[0112] The present disclosure also provides an assay suitable for assessing the autolysis resistance of the recombinant proteases disclosed herein (e.g., Lys-C, Asp-N, and trypsin) and the digestion efficiency of the target protein ("target protein hydrolysis"). In some embodiments, the autolysis resistance of the recombinant protease of the present disclosure is assessed by HPLC. In some embodiments, the autolysis resistance of the recombinant protease of the present disclosure is assessed by MS. In some embodiments, the autolysis resistance of the recombinant protease of the present disclosure is assessed by size exclusion chromatography (SEC). In some embodiments, the autolysis resistance of the recombinant protease of the present disclosure is assessed by HPLC, MS, SEC, HPLC-UV, or any combination thereof. Exemplary, non-limiting methods for testing the autolysis resistance of proteases are described in Example 3 herein.

[0113] Certain combinations of conservative amino acid substitutions and / or chemical modifications to any of the recombinant proteases disclosed herein (e.g., Lys-C, Asp-N, and trypsin) can produce unexpected effects on the proteolytic activity of the enzyme. Therefore, the present disclosure provides methods for determining the effects of the disclosed mutations and / or chemical modifications on the enzymatic activity of the target protein for the protease. In some embodiments, the proteolytic efficacy of the recombinant protease of the present disclosure to the target protein is assessed by MS. In some embodiments, the proteolytic efficacy of the recombinant protease of the present disclosure to the target protein is assessed by size exclusion chromatography (SEC). In some embodiments, the proteolytic efficacy of the recombinant protease of the present disclosure to the target protein is assessed by HPLC, MS, SEC, HPLC-UV, or any combination thereof. Exemplary, non-limiting methods for testing the proteolytic efficacy of protease are described in Example 4 herein.

[0114] How to use

[0115] The present disclosure provides methods for using the disclosed recombinant proteases in a variety of applications. As described above, proteases with enhanced autolysis resistance are particularly useful for analytical methods for analyzing proteins, including HPLC and / or MS. Autolysis produces undesirable peptide fragments from the protease itself during the proteolysis of the target analyte, resulting in interference peaks during HPLC or MS separations, thereby confusing the peptide peaks corresponding to the analyte of interest. Therefore, autolysis-resistant proteases advantageously minimize such interference peaks and improve the sensitivity and specificity of HPLC and / or MS measurements.

[0116] In addition, the disclosed autolysis-resistant proteases are well suited for use in a variety of other applications, including HPLC-UV, development of cell and tissue culture protocols, protein degradation, protein sequencing, dissociation of adherent cells, analysis of protein-protein interactions, capillary electrophoresis (CE), gel electrophoresis (GE), matrix-assisted laser desorption / ionization (MALDI), hydrogen-deuterium exchange, peptide mapping by electrophoresis, western blotting, protein nuclear magnetic resonance (NMR), protein footprinting, affinity purification, protein imaging, proteomic analysis, and protein conformational studies.

[0117] In addition, the disclosed protease can be used in combination with a method for digesting and analyzing protein therapeutics, viral vector proteomes and protein compositions of T cells and CAR-T cell therapies. For example, recombinant proteins are often used in biotherapeutic applications, and their properties and modifications (such as purity, amino acid sequence, post-translational modification, mutation, etc.) are generally characterized, using MS (see, for example, Fung et al., Bioanalysis 8: 847-56, 2016) and other techniques, including HPLC, SEC and HPLC-UV. As discussed herein, such analytical techniques are highly sensitive to autolysis byproducts, and therefore will benefit from the use of autolysis-resistant proteases, which minimize the contamination of analyte samples with irrelevant and destructive peptide peaks.

[0118] Reagent test kit

[0119] The compositions described herein can be provided in a kit for any practical application described herein. The composition can include one or more recombinant proteases disclosed herein in a suitable container device. In some embodiments, the container device is any suitable container for holding, for example, a liquid or a lyophilized composition, including but not limited to a vial, a test tube, an ampoule, a bottle or a syringe. The syringe holds any volume of liquid suitable for injection into a subject, including but not limited to 0.5cc, 1cc, 2cc, 5cc, 10cc or more. In some embodiments, such containers include injection-molded and / or blow-molded plastic containers, in which the required vials are retained. In some embodiments, the kit also includes printed materials for the materials in the kit. In some embodiments, such containers include injection-molded and / or blow-molded plastic containers, in which the required vials are retained. In some embodiments, the kit also includes printed materials for the materials in the kit. In addition, in some embodiments, the preparation contains a stabilizer to increase the shelf life of the kit, and includes, for example, bovine serum albumin (BSA). In some embodiments, when the composition is a lyophilized composition, the kit also contains a solution preparation to reconstitute the lyophilized preparation. Acceptable reconstitution solutions are well known in the art, including, for example, phosphate buffered saline (PBS).

[0120] The term "packaging material" refers to the physical structure of the components of the test kit. In some embodiments, the packaging material keeps the components sterile and is made of materials (e.g., paper, corrugated fiber, glass, plastic, foil, ampoule, etc.) that are commonly used for such purposes. In some embodiments, the label or package insert includes appropriate written instructions (e.g., instructing the user of the test kit to perform one or more methods disclosed herein). In some embodiments, the test kit also includes labels or instructions for using the test kit components in any method disclosed herein. In some embodiments, the test kit includes the compound in the package or dispenser together with the instructions for applying the compound in the method described herein. In some embodiments, the instructions are on, for example, "printed matter", in the test kit or on paper or cardboard attached thereto, or on a label attached to the test kit or packaging material, or attached to a vial or tube containing the test kit components. In some embodiments, the instructions are also included on a computer-readable medium, such as, for example, CD-ROM, DVD, flash memory device, solid-state memory, magnetic disk and disk device, magnetic tape, cloud computing system and server, etc. In some cases, the program and instructions are permanently, substantially permanently, semi-permanently or non-transiently encoded on the medium.

[0121] Example

[0122] The following examples are put forth to provide one of ordinary skill in the art with a description of how the compositions and methods described herein may be used, prepared, and evaluated, and are intended to be purely illustrative of the disclosure and are not intended to limit the scope of what the inventors regard as their disclosure.

[0123] Example 1: Vector transformation and recombinant protein expression and purification

[0124] The nucleotide sequences encoding each of Lys-C (e.g., SEQ ID NO: 2 or SEQ ID NO: 2), Asp-N (e.g., SEQ ID NO: 6) and trypsin (e.g., SEQ ID NO: 9) are cloned into a protein expression vector such as pET20 or pET21b containing an affinity tag (e.g., His tag) by standard molecular cloning procedures. The sequence accuracy of the recombinant plasmid is verified by DNA sequencing methods and transferred into a suitable Escherichia coli (E. coli) strain by electroporation or chemical-based transformation. Expression of the recombinant protein is induced at an appropriate growth stage of the bacterial host. The expressed protein is purified by affinity chromatography and its quality is analyzed by denaturing polyacrylamide gel (SDS-PAGE) and / or liquid chromatography-mass spectrometry (LC-MS). The list of the expressed recombinant plasmids and proteins is as follows:

[0125] (i) pET20b-LysC_KtoR mt: a recombinant plasmid capable of expressing an engineered LysC mutant (SEQ ID NO: 2) that is resistant to autolysis. Lys-C contains a Lys→Arg mutation.

[0126] (ii) pET20b-AspN_DtoE mt: a recombinant plasmid capable of expressing an engineered AspN mutant (SEQ ID NO: 6) that is resistant to autolysis. Asp-N contains an Asp→Glu mutation.

[0127] (iii) pET20b-Trp_RtoK mt: a recombinant plasmid capable of expressing a mutated form of porcine trypsin for subsequent reductive methylation of Lys residues to render it highly resistant to autolysis. Trypsin contains an Arg→Lys mutation.

[0128] Example 2: Alkylation of proteases

[0129] Reductive methylation of trypsin: After purification of the recombinant protein, the trypsin construct was methylated at lysine residues (Heissel et al., PLoS ONE 14:e0218374, 2019) to improve its resistance to autolysis. The protein was diluted to 1 mg / mL with triethylammonium bicarbonate buffer (pH 8.5; 50 mM) and washed with 2.2 μL 36% formaldehyde and 20 μL sodium cyanoborohydride (NaBH 3 CN; 0.6M) / mg trypsin, treated at room temperature for 10 minutes. The reaction process is optionally carried out at a higher pH to minimize autolysis during the alkylation process. Optionally, the reaction is also carried out in the presence of trypsin inhibitors such as benzamidine. After reductive methylation, trypsin is optionally purified on benzamidine-Sepharose 4b beads. In brief, the beads (100 μL / mg trypsin) are pretreated with 3 volumes of ammonium bicarbonate buffer (pH 8, 50mM) and centrifuged at 5°C. After discarding the supernatant, in the presence of a loading buffer (e.g., saturated L-phenylalanine in 50mM ammonium bicarbonate), the trypsin solution is combined with the beads for 30 minutes by gentle rotation-based mixing. The beads bound to the protein are precipitated, washed with 3 volumes of ammonium bicarbonate buffer (50mM, 3X), and the supernatant is removed by centrifugation. Methylated trypsin is eluted with 12mM HCl by gentle rotation at room temperature. As an alternative to purification by benzamidine Sepharose 4B adsorbent, cold acetone precipitation and centrifugation were used to purify trypsin. The degree of derivatization was measured by LC-MS.

[0130] Chemical derivatization of acidic amino acid residues in Asp-N: It is not necessary to process mutated LysC or AspN variants. However, if it is desired to use wild-type AspN alone or in combination with one or more engineered proteases of the present disclosure, chemical derivatization of acidic amino acid substrates can be advantageous. These acidic residues targeted proteases can be derivatized with carbodiimide in the presence of excessively high concentrations of amines (including but not limited to ethanolamine). In this way, the carboxylic acid groups of the acidic amino acid side chains are converted into neutral amides without causing any significant amount of protein cross-linking (Graceffa et al., Arch Biochem Biophys. 297: 46-51, 1992). Because the reaction products containing amides cannot act as substrates, autolysis is further minimized. The method involves treating the purified protein solution at room temperature for 2-3 hours with 0.2-0.5M ethanolamine (pH 4.0, from 1-2M stock solution), 30mM Mes buffer (pH 5.5, from 0.5M stock solution), and 12mM 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) (from 100mM stock solution) in 40mM NaCl, 5mM MOPS, 0.2mM EDTA, pH 7.5, 0.2mM DTT and a protease inhibitor cocktail (0.25mM PMSF, 0.75mM benzamidine, 1μg / mL leupeptin), and quenching with 0.1M-0.2M β-mercaptoethanol. The obtained engineered protease was tested for autolysis resistance as described in Example 3 below. The degree of derivatization was measured by LC-MS.

[0131] Example 3: Autolysis Assay

[0132] The autodigestion of the purified engineered protease was monitored by incubating a defined amount of protease in 50 mM ammonium bicarbonate buffer at 37°C for 10 minutes to 16 hours and analyzing the resulting solution by reverse phase high performance liquid chromatography (RP-HPLC) using gradient chromatography. The mobile phase for this analysis included water (A) and acetonitrile (B) acidified with 0.1% trifluoroacetic acid (TFA). Difluoroacetic acid or formic acid was also used as a mobile phase additive with other types of RP columns. The percentage of intact protein and autolytic peptides was evaluated by LC-UV or LC-MS analysis. Alternatively, size exclusion chromatography can be applied to determine the intact protease versus the autolytic protease.

[0133] Example 4: Protocol for testing the digestion efficiency of engineered proteases

[0134] The target protein, such as NIST mAb reference material 8671 or a small protein, such as lysozyme or cytochrome C (10 μg in 10 μL), was denatured with 6 M guanidine hydrochloride (90 μL) and treated with dithiothreitol (2 μL, 250 mM) for 30 minutes at room temperature to reduce disulfide bonds. The reduced form of cysteine ​​was alkylated with iodoacetamide (3 μL, 350 mM) in the dark at room temperature for 30 minutes. Subsequently, the protein was desalted on gel filtration by gravity or spin columns using digestion buffer (100 mM Tris pH 7.5), the concentration was adjusted to 0.2 μg / μL, and digested with protease (1:5 or 1:20 ratio) at 37°C for 60 minutes. The reaction was quenched with 1% formic acid (20 μL) and stored at -20°C for subsequent LC-MS analysis.

[0135] ACQUITY Premier Peptide CSH C18 column ( 1.7 μm, 2.1×150 mm), using 65°C column temperature and water and acetonitrile mobile phase modified with 0.1% formic acid or 0.05% difluoroacetic acid. A flow rate of 0.25 mL / min was applied. The gradient conditions were programmed to hold 1% B solution for 5 minutes, change to 40% B solution after 65 minutes, change to 70% B solution after 3 minutes, then hold for 2 minutes, then switch to re-equilibrium conditions (1% B solution), and hold for 15 minutes.

[0136] Mass spectrometry data were obtained in full scan mode using a time-of-flight mass spectrometer operating in positive ion mode at 2 Hz with a scan range of 50 m / z-2000 m / z. When using a BioAccord instrument containing a benchtop ToF mass spectrometer, the electrospray source conditions were programmed to a desolvation temperature of 350°C, a cone voltage of 20 V, and a capillary voltage of 1.2 kV. In MSe mode, fragmentation data were acquired by adjusting the cone voltage to a range of 60 V-120 V.

[0137] Other Implementations

[0138] Various modifications and variations of the disclosed embodiments described will be apparent to those skilled in the art without departing from the scope and spirit of the present disclosure. Although the present disclosure has been described in conjunction with specific embodiments, it should be understood that the present disclosure protected by the claims should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the present disclosure that are apparent to those skilled in the art are intended to be within the scope of the present disclosure. Other embodiments are protected by the claims.

Claims

1. A recombinant protease comprising one or more conservative amino acid substitutions, wherein the conservative amino acid substitutions enhance the autolysis resistance of the protease compared to the autolysis resistance of the protease in the absence of the one or more conservative substitutions, wherein the protease is an endopeptidase Lys-C (Lys-C).

2. A recombinant protease comprising one or more conservative amino acid substitutions, which enhance the autolysis resistance of the protease compared to the autolysis resistance of the protease in the absence of the one or more conservative substitutions, wherein the protease is Asp-N protease (Asp-N).

3. A recombinant protease comprising one or more conservative amino acid substitutions, which enhance the autolysis resistance of the trypsin compared to the autolysis resistance of the trypsin in the absence of the one or more conservative substitutions, wherein the protease is trypsin.

4. The recombinant protease of any one of claims 1-3, wherein the one or more conservative substitutions comprise at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, or at least 15 conservative substitutions.

5. The recombinant protease of claim 4, wherein the one or more conservative amino acid substitutions comprise 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 conservative substitutions.

6. The recombinant protease according to any one of claims 1, 4 or 5, wherein the one or more conservative substitutions are substitutions of lysine (Lys) to arginine (Arg).

7. The recombinant protease of claim 6, wherein the one or more conservative substitutions are selected from residues 2, 39, 52, 54, 62, 104, 173, 178, 183, 205, 235, 254, 311, 360, and 408 of SEQ ID NO: 1 or residues 30, Lys to Arg substitution at one or more amino acid residues 49, 106, 155 and 203.

8. The recombinant protease according to any one of claims 2, 4 or 5, wherein at least one or more conservative substitutions are substitutions of aspartic acid (Asp) to glutamic acid (Glu).

9. The recombinant protease of claim 8, wherein the one or more conservative substitutions comprise an Asp to Glu substitution at one or more amino acid residues selected from residues 2, 14, 46, and 130 of SEQ ID NO:

5.

10. The recombinant protease of claim 9, wherein the one or more conservative substitutions further comprise an Asp to Glu substitution at one or more of amino acid residues 67 and 71 of SEQ ID NO:

5.

11. The recombinant protease of any one of claims 3-5, wherein the one or more conservative substitutions are substitutions from Arg to Lys.

12. The recombinant protease of claim 11, wherein the one or more conservative substitutions comprise an Arg to Lys substitution at one or more amino acid residues selected from residues 45, 49, 99 and 107 of SEQ ID NO:

9.

13. The recombinant protease of any one of claims 1-12, wherein the autolysis resistance of the protease is enhanced by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% compared to the autolysis resistance of the protease in the absence of the one or more conservative substitutions.

14. The recombinant protease of any one of claims 1-13, wherein the protease comprises one or more chemical modifications.

15. The recombinant protease of claim 14, wherein the one or more chemical modifications comprise an alkyl moiety, an acetyl moiety, an amide moiety, an ester moiety, an imine moiety, an amidino moiety, a guanidino moiety, or a thioether moiety.

16. The recombinant protease of claim 15, wherein the alkyl moiety is selected from the group consisting of a methyl moiety, a dimethyl moiety, an octanal moiety, and a cyclodextrin monoaldehyde moiety.

17. The recombinant protease of any one of claims 1-16, wherein the protease is porcine, bovine, rat, murine, human, avian, bacterial, fungal, plant, artificial or synthetic.

18. The recombinant protease according to any one of claims 1 to 17, wherein the active site of the protease does not contain an amino acid substitution.

19. The recombinant protease of any one of claims 1, 4-7, or 13-18, wherein the protease comprises the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO:

4.

20. The recombinant protease of any one of claims 2, 4, 5, 8, 9, 10, or 13-14, wherein the protease comprises the amino acid sequence of SEQ ID NO: 6 or SEQ ID NO:

7.

21. The recombinant protease of any one of claims 3, 4, 5, or 11-20, wherein the protease comprises the amino acid sequence of SEQ ID NO:

9.

22. A method of reducing the level of peptide by-products of protease autolysis in an analytical assay, the method comprising using a recombinant protease according to any one of claims 1-21.

23. The method of claim 22, wherein the analytical assay is selected from liquid chromatography (LC), LC-mass spectrometry (LC-MS), LC-UV, capillary electrophoresis (CE), gel electrophoresis (GE), and matrix-assisted laser desorption / ionization (MALDI).

24. The method of claim 23, wherein the analytical assay is LC-MS.

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