Chymotrypsinogen and use thereof

Abstract

Provided are a chymotrypsinogen and the use thereof. The present invention specifically relates to a protein tag capable of improving the activation efficiency of a recombinant chymotrypsinogen. Compared with naturally extracted human chymotrypsinogen, the recombinant chymotrypsinogen in which the protein tag is fused has a significantly improved activation efficiency. The present invention also relates to a recombinant chymotrypsinogen comprising the protein tag, a nucleic acid molecule encoding the recombinant chymotrypsinogen, a vector comprising the nucleotide sequence of the nucleic acid molecule, a nucleic acid construct and a host cell. In addition, the present invention also relates to a method for expressing the recombinant chymotrypsinogen.

Description

Chymotrypsinogen and its uses

[0001] Priority Statement

[0002] This disclosure claims priority to the following patent applications:

[0003] The Chinese patent application, filed on October 16, 2024, with application number 2024114509542, and entitled "chymotrypsinogen and its uses", is titled "chymotrypsinogen and its uses".

[0004] The Chinese patent application, filed on December 27, 2024, with application number 202411955301X, and entitled "Chymotrypsinogen and its Uses", is titled "Chymotrypsinogen and its Uses".

[0005] This disclosure incorporates the full text of the aforementioned Chinese patent application. Technical Field

[0006] This disclosure relates to the field of biotechnology, specifically to a chymotrypsinogen and its uses. Background Technology

[0007] chymotrypsin (EC3.4.21.1), also known as chymotrypsin, belongs to the serine protease family. It is produced in vivo as a zymogen and is hydrolyzed by trypsin and itself to form active chymotrypsin. It mainly hydrolyzes peptide chains with large hydrophobic amino acid residues at the carboxyl terminus, such as Tyr, Trp, Phe, and Leu.

[0008] The use of chymotrypsin in clinical practice is very extensive. It is mainly used for surgical treatments such as inflammation, inflammatory edema, hematoma, adhesions, and ulcers after surgery or trauma; thoracic treatments such as empyema, hemothorax, postoperative difficulty in expectoration, and lung distension; internal medicine treatments such as chronic bronchitis and bronchial asthma; obstetric and gynecological treatments such as cervicitis, salpingitis, and pelvic inflammatory disease; and ophthalmological and ENT treatments such as keratitis and suppurative otitis media.

[0009] Currently, chymotrypsin is mainly extracted from the pancreas of pigs and cattle. Due to its animal origin, it carries the risk of contamination by unknown viruses and exogenous factors, significantly limiting its application in the pharmaceutical industry. Furthermore, because trypsin and chymotrypsin have similar structures and properties, extraction, separation, and purification of raw materials cannot yield completely isolated chymotrypsin, and even obtaining high-purity chymotrypsin is difficult. High-purity human chymotrypsinogen can be produced by expressing human chymotrypsinogen through recombinant engineered bacteria; the zymogen can then be activated to obtain human chymotrypsin. Replacing chymotrypsin extracted from animal pancreas with animal-free human chymotrypsin aligns with the development trend of biopharmaceuticals, and large-scale production of human chymotrypsin is needed in this field.

[0010] Animal-derived chymotrypsins, such as bovine chymotrypsinogen, are secreted by the pancreas. After entering the small intestine with pancreatic juice, they are cleaved by trypsin between Arg 15 and Ile 16 (sequence numbered according to Bovine chymotrypsin A) into two parts linked by disulfide bonds. Subsequently, the short peptide Ser 14-Arg 15 is cleaved at Leu 13, and the short peptide Tyr147-Asn 148 is cleaved at Tyr 146 and Asn 148, forming a biologically active chymotrypsin consisting of three disulfide-linked peptide chains. This is an essential step in the production of chymotrypsin using recombinant engineered bacteria. However, whether the activation process of human chymotrypsin is the same as that of animal-derived chymotrypsin remains to be verified.

[0011] On the other hand, the activation time of naturally extracted chymotrypsinogen is relatively long, often requiring more than 12 hours. Reducing the activation time of chymotrypsinogen could shorten the production cycle and save production costs. Therefore, there is an urgent need in this field for a novel chymotrypsinogen with higher activation efficiency, thereby improving the production efficiency of chymotrypsin. Summary of the Invention

[0012] This disclosure solves the above-mentioned technical problems existing in the prior art through the following technical solutions: On the one hand, the inventors of this disclosure discovered that human chymotrypsin and animal chymotrypsin have different subunit structures. Unlike the three-chain structure of animal chymotrypsin, human chymotrypsinogen is enzymatically cleaved into two chains, thereby forming human chymotrypsin.

[0013] On the other hand, this disclosure provides a protein tag that, when fused with naturally extracted chymotrypsinogen, yields a recombinant chymotrypsinogen that significantly reduces the activation time of the recombinant chymotrypsinogen compared to the naturally extracted chymotrypsinogen. Furthermore, the recombinant chymotrypsinogen, after activation, exhibits a specific activity that is substantially the same as or higher than that of the naturally extracted chymotrypsin, thereby effectively promoting the production of chymotrypsin.

[0014] The technical solution provided in this disclosure is as follows:

[0015] In a first aspect, this disclosure provides a human chymotrypsin, wherein the chymotrypsin is composed of an A chain and a B chain linked by disulfide bonds; the amino acid sequence of the A chain has at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO:5; and the amino acid sequence of the B chain has at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO:6.

[0016] In an optional embodiment, chain A comprises the amino acid sequence shown in SEQ ID NO:5; and chain B comprises the amino acid sequence shown in SEQ ID NO:6. For example, the amino acid sequence of chain A is as shown in SEQ ID NO:5; and the amino acid sequence of chain B is as shown in SEQ ID NO:6.

[0017] In an optional embodiment, the B chain is connected to the A chain via a disulfide bond formed by C27, C43, C107, C121, C153, C167, C176, C186 or C205; alternatively, the B chain is connected to the A chain via a disulfide bond formed by C107.

[0018] Optionally, the B chain further contains at least one intramolecular disulfide bond selected from C27-C43, C121-C186, C153-C167, or C176-C205;

[0019] Optionally, the B chain also contains intramolecular disulfide bonds C27-C43, C121-C186, C153-C167 and C176-C205.

[0020] In an optional embodiment, the specific activity of the human chymotrypsin is greater than 1000 units / mg.

[0021] In an optional embodiment, the human chymotrypsin is a recombinant human chymotrypsin.

[0022] A second aspect of this disclosure provides the use of the protein tag EAEA in (a) or (b):

[0023] (a) Improves the activation efficiency of chymotrypsinogen;

[0024] (b) Fusing with chymotrypsinogen to prepare recombinant chymotrypsinogen containing the protein tag EAEA, wherein the recombinant chymotrypsinogen containing the protein tag EAEA has a higher activation efficiency than chymotrypsinogen without the protein tag EAEA.

[0025] In an optional embodiment, the protein tag EAEA is located at the N-terminus of the recombinant chymotrypsinogen.

[0026] In an optional embodiment, the activation time of the recombinant chymotrypsinogen containing the protein tag EAEA is reduced to less than 80%, 70%, 60%, 50%, 40%, 30%, or 20% of the activation time of the chymotrypsinogen without the protein tag EAEA; optionally, the chymotrypsin obtained by activating the recombinant chymotrypsinogen containing the protein tag EAEA has substantially the same or higher specific activity compared to the chymotrypsin obtained by activating the chymotrypsinogen without the protein tag EAEA.

[0027] In an optional embodiment, the chymotrypsinogen is a human chymotrypsinogen.

[0028] In an optional embodiment, the amino acid sequence of the chymotrypsinogen has at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO:1.

[0029] A third aspect of this disclosure provides a recombinant chymotrypsinogen with the protein tag EAEA linked to its N-terminus.

[0030] In an optional embodiment, the activation time of the recombinant chymotrypsinogen is reduced to less than 80%, less than 70%, less than 60%, less than 50%, less than 40%, less than 30%, or less than 20% compared to chymotrypsinogen without the protein tag EAEA; optionally, the chymotrypsin obtained by activating the recombinant chymotrypsinogen has substantially the same or higher specific activity compared to the chymotrypsin obtained by activating chymotrypsinogen without the protein tag EAEA.

[0031] In an optional embodiment, the chymotrypsinogen is a human chymotrypsinogen.

[0032] In an optional embodiment, the amino acid sequence of the chymotrypsinogen has at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO:2.

[0033] The fourth aspect of this disclosure provides a nucleic acid molecule encoding the recombinant chymotrypsinogen described in any of the foregoing embodiments; optionally, the nucleotide sequence encoding the protein tag EAEA is GAGGCTGAAGCT (SEQ ID NO:10).

[0034] The fifth aspect of this disclosure provides a recombinant expression vector comprising the nucleotide sequence of the nucleic acid molecule described in the foregoing embodiments.

[0035] The sixth aspect of this disclosure provides a recombinant host cell comprising the nucleic acid molecule or the recombinant expression vector described in the foregoing embodiments; optionally, the host cell is selected from bacterial cells or fungal cells, such as Escherichia coli cells or Pichia pastoris cells.

[0036] The seventh aspect of this disclosure provides a method for preparing recombinant chymotrypsinogen according to any embodiment of the third aspect, comprising the following steps:

[0037] (a) The recombinant host cells described in the sixth aspect are cultured under conditions that favor the expression of the recombinant chymotrypsinogen as described in any embodiment of the third aspect;

[0038] (b) Recovery of the expressed recombinant chymotrypsinogen from the culture medium.

[0039] The eighth aspect of this disclosure provides a method for preparing recombinant chymotrypsin, comprising activating the recombinant chymotrypsinogen obtained in any embodiment of the third aspect or the recombinant chymotrypsin prepared by the method for preparing recombinant chymotrypsinogen described in the seventh aspect with a chymotrypsinogen activator to obtain recombinant chymotrypsin.

[0040] Optionally, the chymotrypsinogen activator is trypsin.

[0041] A ninth aspect of this disclosure provides another method for preparing recombinant chymotrypsin, comprising preparing the human chymotrypsin A chain and the human chymotrypsin B chain separately, and then assembling the A chain and the B chain to obtain recombinant chymotrypsin.

[0042] Optionally, the A chain can be synthesized by chemical methods, and the B chain can be prepared by gene recombination methods.

[0043] The tenth aspect of this disclosure provides a composition or kit comprising: (i) the recombinant chymotrypsinogen as described in any of the preceding embodiments; and (ii) an optional pharmaceutically acceptable carrier or excipient.

[0044] In an optional embodiment, the composition or kit further comprises a chymotrypsinogen activator; optionally, the chymotrypsinogen activator is trypsin.

[0045] The eleventh aspect of this disclosure provides a pharmaceutical composition comprising (a) a therapeutically effective amount of human chymotrypsin or recombinant human chymotrypsin as described in any of the preceding embodiments, and (b) a pharmaceutically acceptable carrier.

[0046] The human chymotrypsin disclosed herein has a double-stranded structure. Furthermore, this disclosure also provides a protein tag for improving the activation efficiency of chymotrypsinogen, and a method for preparing chymotrypsinogen with high activation efficiency, and uses the protein tag and the preparation method to prepare a double-stranded human chymotrypsin.

[0047] To make the technical solutions described in this disclosure clearer, the following description, in conjunction with the accompanying drawings and specific embodiments, will provide further details. Attached Figure Description

[0048] Figure 1 shows the electrophoresis results of the expression products of the two chymotrypsinogen clones.

[0049] Figure 2 shows the activation curves of the two chymotrypsinogens.

[0050] Figure 3 shows the reduced mass spectrum after hCTRB activation.

[0051] Figure 4 shows the reduced mass spectrum after EAEA-hCTRB activation.

[0052] Invention Details

[0053] I. Definition

[0054] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. While any methods, apparatus, and materials similar to or equivalent to those described herein may be used in the practice or testing of this disclosure, preferred methods, apparatus, and materials are now described.

[0055] When used herein, the term "recombinant human chymotrypsinogen" refers to a protein sequence or functional fragment thereof obtained through genetic engineering recombination techniques from naturally extracted human chymotrypsinogen. "Recombinant human chymotrypsinogen" includes variant polypeptides with one or more amino acid substitutions, deletions, or insertions relative to the sequence of SEQ ID NO:1. When used herein, "recombinant human chymotrypsinogen" can be activated to chymotrypsin at a significantly shorter activation time than naturally extracted human chymotrypsinogen (e.g., below 80%, 70%, 60%, 50%, 40%, 30%, or 25%), and / or, when activated to chymotrypsin, has a specific activity (U / mg) substantially the same as or higher than that of naturally extracted human chymotrypsin. "Chymotrypsin" is commonly used for peptide bond hydrolysis, belongs to the class of endopeptidases, has a high hydrolysis yield, and its function and uses are similar to trypsin, but it has a stronger degrading ability, lower toxicity, and fewer adverse reactions than trypsin. Trypsin has a wide range of uses, including the treatment of sprains, otitis media, rhinitis, sinusitis, pharyngitis, and lung abscesses. It can also be used for surgical inflammation, trauma, hematomas, and abscesses, and is performed during tracheotomy. Trypsin is particularly effective for patients with excessive phlegm, making it easier to cough up.

[0056] The term "disulfide bond" as used in this article refers to a single covalent bond between two sulfur elements (-SS-), which in biochemistry is formed between cysteine ​​residues in two proteins or between two cysteine ​​residues within a protein. Sulfur bonding plays a crucial role in determining the secondary and tertiary structure of proteins.

[0057] The terms “disulfide bond” and “disulfide bond formation” refer to intramolecular disulfide bonds and the formation of disulfide bonds, unless otherwise specified.

[0058] The terms “intramolecular disulfide bond” and “formation of intramolecular disulfide bond” refer to intramolecular disulfide bonds that are unique to natural proteins, unless otherwise specified.

[0059] The terms "nucleotide sequence" or "polynucleotide" refer to deoxyribonucleotides, deoxyribonucleosides, ribonucleosides, or ribonucleotides and their polymers in single-stranded or double-stranded form. Unless specifically limited, the term encompasses nucleic acids containing known analogs of natural nucleotides, which have binding properties similar to a reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise specifically limited, the term also refers to oligonucleotide analogs, including PNAs (peptide nucleic acids), DNA analogs (phosphate thioesters, phosphoramidites, etc.) used in antisense techniques. Unless otherwise specified, a specific nucleic acid sequence also implicitly encompasses variants of its conserved modifications (including, but not limited to, degenerate codon substitutions) and complementary sequences, as well as explicitly specified sequences. Specifically, degenerate codon substitution can be achieved by generating a sequence in which the 3rd position of one or more selected (or all) codons is substituted with a mixed base and / or deoxyinosine residue. The polynucleotide encoding the "recombinant human chymotrypsinogen" of this disclosure comprises variant nucleic acids having one or more nucleotide substitutions, deletions, or insertions relative to the sequence SEQ ID NO:4.

[0060] The terms “peptide,” “protein,” or “protein protein” are used interchangeably herein to refer to a polymer of amino acid residues. That is, the description of a peptide is equally applicable to the description of a peptide and the description of a protein, and vice versa. The terminology applies to naturally occurring amino acid polymers as well as amino acid polymers in which one or more amino acid residues are non-naturally encoded amino acids. As used herein, the terminology covers amino acid chains of any length, including full-length proteins, wherein the amino acid residues are linked by covalent peptide bonds.

[0061] The term "sequence identity" describes the correlation between two nucleotide sequences.

[0062] For the purposes of this disclosure, the degree of sequence identity between two amino acid sequences was determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J.Mol.Biol.48:443-453) executed in the Needle program, preferably version 3.0.0 or later, such as the EMBOSS software package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends in Genetics 16:276-277), preferably version 3.0.0 or later. Optional parameters used were a gap open penalty of 10, a gap extension penalty of 0.5, and an EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix. The percentage identity was calculated using the Needle output labeled "longest identity" (obtained using the -nobrief option) and calculated as follows:

[0063] (Same residues × 100) / (Alignment length - total number of gaps in alignment)

[0064] For the purposes of this disclosure, the degree of sequence identity between two deoxyribonucleotide sequences was determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, see above) executed in the Needle program, preferably version 3.0.0 or higher, such as EMBOSS software (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, see above). Optional parameters used were a nick opening penalty of 10, a nick extension penalty of 0.5, and an EDNAFULL (EMBOSS version of NCBI NUC4.4) substitution matrix. The percentage identity was calculated using the Needle output labeled “Highest Identity” (obtained using the -nobrief option), and was calculated as follows:

[0065] (same deoxyribonucleotides × 100) / (alignment length - total number of gaps in alignment)

[0066] The term "nucleic acid construct" refers to single-stranded or double-stranded nucleic acid molecules that are isolated from naturally occurring genes, modified to contain segments of nucleic acid in a manner not otherwise existing in nature, or synthesized.

[0067] The term "regulatory sequence" is defined as all components included in the methods of this disclosure that are essential for polynucleotide expression. Each regulatory sequence may be native or heterologous to the polynucleotide sequence, or each regulatory sequence may be native or heterologous to each other. These regulatory sequences include, but are not limited to, polyadenylated sequences, propeptide sequences, promoters, signal peptide sequences, and transcription terminators. At a minimum, the regulatory sequence includes a promoter and a termination signal for transcription and translation. The regulatory sequence may be provided together with a linker for introducing a specific restriction site that facilitates the connection of the regulatory sequence to the coding region of the nucleotide sequence encoding the polypeptide.

[0068] The term "operably linked" in this document refers to a configuration in which a regulatory sequence is placed in the appropriate position relative to the coding sequence of a polynucleotide sequence, such that the regulatory sequence guides the expression of the coding sequence of the polypeptide.

[0069] The term “expression” includes any step involved in the production of a polypeptide, including but not limited to transcription, post-transcriptional modification, translation, post-translational modification, and secretion.

[0070] The term “expression vector” is defined herein as a linear or circular DNA molecule containing a polynucleotide having encoded a polypeptide disclosed herein and operatively linked to additional nucleotides provided for its expression.

[0071] The term "host cell" refers to a cell containing the nucleotides disclosed herein, regardless of the method used for insertion to produce a recombinant host cell, such as direct uptake, transduction, pairing, or other methods known in the art. Exogenous polynucleotides may remain as non-integrating vectors, such as plasmids, or may be integrated into the host genome. Host cells may be prokaryotic or eukaryotic cells.

[0072] The term "conversion" refers to the method of introducing a heterologous DNA sequence into a host cell or organism.

[0073] The term "scale-up" broadly refers to the process of scaling up an apparatus from laboratory scale to industrial-scale production. As used herein, the term includes scaling up from small shake flask expressions to small or larger fermenters.

[0074] The term "pharmaceutically acceptable carrier" refers to an effective amount of the active substance of this application that does not interfere with the biological activity of the active substance and the host or patient, or with the toxicity or side effects of any reagent or carrier medium. Representative carriers include water and mineral oil, cream bases, lotion bases, ointment bases, etc. Such carriers include suspending agents, binders, thickeners, flavoring agents, coloring agents, antioxidants, etc.

[0075] In this disclosure, the term "suspending agent" refers to an additive that increases the viscosity of the dispersion medium to reduce the settling velocity of particles or increase the hydrophilicity of particles. Suspending agents can be classified according to molecular weight into low-molecular-weight suspending agents such as glycerol, syrup, and sorbitol; high-molecular-weight suspending agents such as gums like gum arabic and tragacanth gum; plant mucilages and polysaccharides such as sodium alginate, agar, and starch; and cellulose derivatives such as methylcellulose, sodium carboxymethyl cellulose, and hydroxypropyl cellulose.

[0076] In this disclosure, the term "adhesive" refers to a viscous substance that bonds two separate materials together by virtue of its adhesive properties. In this disclosure, adhesives are used to bind and shape pharmaceutical products. Based on their source, adhesives can be classified as natural adhesives, such as starch, protein, dextrin, animal glue, etc.; and synthetic adhesives.

[0077] In this disclosure, the term "thickener," also known as a gelling agent, refers to a substance that increases the viscosity of latex or liquids. Based on their ionic properties, they can be divided into two main categories: ionic thickeners, such as alginate, sodium carboxymethyl cellulose, and starch; and nonionic thickeners, such as sodium propylene glycol alginate and hydroxypropyl starch.

[0078] In this disclosure, the term "flavoring agent" refers to a pharmaceutical excipient used to improve or mask the unpleasant odor and taste of a drug, making it difficult for the patient to detect the drug's strong bitterness (or other unpleasant tastes such as spiciness, irritation, etc.).

[0079] In this disclosure, the term "coloring agent" refers to a substance that imparts color to a pharmaceutical preparation and improves the color of the pharmaceutical preparation.

[0080] In this disclosure, the term "antioxidant" refers to a substance that prevents the adverse effects of oxygen.

[0081] The term “amino acid” or “amino acid residue” refers to naturally occurring amino acids, non-natural amino acids that function in a similar manner to naturally occurring amino acids, amino acid analogs, and amino acid mimics, which are all stereoisomers if their structure allows for their D and L stereoisomer forms. Amino acids are referred to herein by their names, their well-known three-letter symbols, or the single-letter symbols recommended by the IUPAC-IUB Committee on Biochemistry Nomenclature.

[0082] When used in conjunction with amino acids, the term “naturally occurring” refers to the 20 common amino acids (i.e., alanine (A), cysteine ​​(C), aspartic acid (D), glutamic acid (E), phenylalanine (F), glycine (G), histidine (H), isoleucine (I), lysine (K), leucine (L), methionine (M), asparagine (N), proline (P), glutamine (Q), arginine (R), serine (S), threonine (T), valine (V), tryptophan (W), and tyrosine (Y)), as well as selenocysteine, pyrrolidone (PYL), and pyrrolidone-carboxylysine (PCL).

[0083] As used herein, the term "non-natural amino acid" means an amino acid that is not encoded by the genetic code of any organism or that has not been found in any organism. It can be, for example, a purely synthetic compound. Examples of non-natural amino acids include, but are not limited to, hydroxyproline, γ-carboxyglutamic acid, O-phosphoserine, azetidine carboxylicacid, 2-aminoadipic acid, 3-aminoadipic acid, β-alanine, aminopropionic acid, 2-aminobutyric acid, 4-aminobutyric acid, 6-aminohexanoic acid, 2-aminoheptanoic acid, 2-aminoisobutyric acid (Aib), 3-aminoisobutyric acid, 2-aminopimelic acid, tert-butylglycine, 2,4-diaminoisobutyric acid, desmosine, 2,2'-diaminopimelic acid, 2,3-diaminopropionic acid, and N-ethylglycine. N-methylglycine, N-ethylasparagine, homoproline, hydroxylysine, allo-hydroxylysine, 3-hydroxyproline, 4-hydroxyproline, isodesin, allo-isoleucine, N-methylalanine, N-methylglycine, N-methylisoleucine, N-methylpentylglycine, N-methylvaline, naphthylalanine, valine, leucine, ornithine, D-ornithine, D-arginine, p-aminophenylalanine, pentylglycine, pipecolic acid, and thioproline.

[0084] As used herein, the term "amino acid analogue" refers to a compound having the same basic chemical structure as a naturally occurring amino acid. Amino acid analogues include natural and non-natural amino acids that have been reversibly or irreversibly chemically blocked, or whose C-terminal carboxyl group, N-terminal amino group, and / or side-chain functional groups have been chemically modified. Such analogues include, but are not limited to, methionine sulfoxide, methionine sulfone, S-(carboxymethyl)-cysteine, S-(carboxymethyl)-cysteine ​​sulfoxide, S-(carboxymethyl)-cysteine ​​sulfone, aspartic acid-(β-methyl ester), N-ethylglycine, alanine carboxamide, homoserine, ortholeucine, and methionine methylsulfonium.

[0085] As used herein, the term "amino acid analogue" refers to a chemical compound that has a structure different from the general chemical structure of amino acids, but functions in a manner similar to that of naturally occurring amino acids.

[0086] In some embodiments, the variant includes at least one additional amino acid at its N-terminus. In one embodiment, the at least one additional amino acid is selected from naturally occurring amino acids other than proline, non-natural amino acids, amino acid analogs, and amino acid mimics. In one embodiment, the at least one additional amino acid is selected from G, A, N, and C. In a particular embodiment, the at least one additional amino acid is G.

[0087] As used in this article, the term "D-amino acid," in contrast to L-amino acid, refers to two isomers of the same amino acid with different optical rotations. According to the Fischer projection, L-amino acids have the amino group on the left and D-amino acids have the amino group on the right. Typically, naturally occurring amino acids are L-amino acids, while D-amino acids must be obtained through artificial synthesis.

[0088] As used herein, families of amino acid residues with similar side chains are defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), amino acids with acidic side chains (e.g., aspartic acid, glutamic acid), amino acids with nonpolar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), amino acids with nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), amino acids with β-branched side chains (e.g., threonine, valine, isoleucine), and amino acids with aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).

[0089] Examples of amino acids that can be conservedly substituted for each other are shown in the table below:

[0090] II. The human chymotrypsin disclosed herein

[0091] Unlike animal-derived chymotrypsin, the human-derived chymotrypsin provided in this disclosure consists of an A chain and a B chain linked by disulfide bonds; the amino acid sequence of the A chain has at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO:5; the amino acid sequence of the B chain has at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO:6.

[0092] The amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity may be obtained by conservative substitution of one or more amino acid residues in the amino acid sequences shown in SEQ ID NO:5 and / or SEQ ID NO:6. Alternatively, it may be obtained by non-conservative substitution of one or more amino acid residues in the amino acid sequences shown in SEQ ID NO:5 and / or SEQ ID NO:6, while still retaining the chymotrypsin function. Or, it may be obtained by adding or deleting one or more amino acid residues based on the amino acid sequences shown in SEQ ID NO:5, SEQ ID NO:6, and / or SEQ ID NO:1, while still retaining the chymotrypsin function.

[0093] For example, chain A comprises the amino acid sequence shown in SEQ ID NO:5; chain B comprises the amino acid sequence shown in SEQ ID NO:6, and the function of the human chymotrypsin is not affected. Alternatively, the amino acid sequence of chain A is as shown in SEQ ID NO:5; the amino acid sequence of chain B is as shown in SEQ ID NO:6. Alternatively, the amino acid sequence of the human chymotrypsin is as shown in SEQ ID NO:1.

[0094] In one embodiment, the B chain is connected to the A chain via a disulfide bond formed by C27, C43, C107, C121, C153, C167, C176, C186 or C205; optionally, the B chain is connected to the A chain via a disulfide bond formed by C107.

[0095] Optionally, the B chain further contains at least one intramolecular disulfide bond selected from C27-C43, C121-C186, C153-C167, or C176-C205.

[0096] Optionally, the B chain also contains intramolecular disulfide bonds C27-C43, C121-C186, C153-C167 and C176-C205.

[0097] In one embodiment, the specific activity of the human chymotrypsin is greater than 1000 units / mg, including but not limited to 1000 units / mg, 1200 units / mg, 1400 units / mg, 1600 units / mg, 1800 units / mg or 2000 units / mg.

[0098] In one embodiment, the N-terminus of the A chain of the human chymotrypsin may further contain a protein tag EAEA, i.e., recombinant human chymotrypsin. The amino acid sequence of the A chain of the recombinant human chymotrypsin has at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO:7.

[0099] III. This disclosure provides protein tagging.

[0100] This disclosure provides a protein tag with the amino acid sequence EAEA, which can be used to fuse with chymotrypsinogen to construct recombinant chymotrypsinogen. The recombinant chymotrypsinogen has a higher activation efficiency than naturally extracted chymotrypsinogen. When the activation time of chymotrypsinogen is used as an evaluation indicator, the activation time of the recombinant chymotrypsinogen is reduced to less than 80%, 70%, 60%, 50%, 40%, 30%, or 20% of the activation time of naturally extracted chymotrypsinogen. Simultaneously, the chymotrypsin obtained by activating the recombinant chymotrypsinogen has substantially the same or higher specific activity compared to the chymotrypsin obtained by activating the naturally extracted chymotrypsinogen.

[0101] In one embodiment, the chymotrypsinogen is a human chymotrypsinogen.

[0102] Based on the aforementioned technical effects, the protein tag EAEA provided in this disclosure can be used to (a) improve the activation efficiency of chymotrypsinogen; or (b) fuse with naturally extracted chymotrypsinogen to prepare recombinant chymotrypsinogen, wherein the activation efficiency of the recombinant chymotrypsinogen is higher than that of the naturally extracted chymotrypsinogen. Optionally, it can be used to fuse with human chymotrypsinogen, for example, the amino acid sequence of the naturally extracted human chymotrypsinogen having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO:1.

[0103] IV. The recombinant human chymotrypsinogen disclosed herein

[0104] This disclosure provides a recombinant human chymotrypsinogen, wherein the activation efficiency of the recombinant human chymotrypsinogen is significantly higher than that of naturally extracted human chymotrypsinogen. In one embodiment, the recombinant human chymotrypsinogen of this disclosure can be activated into chymotrypsin at activation times of less than 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, or 25% of the activation time of naturally extracted human chymotrypsinogen. In one embodiment, the recombinant human chymotrypsinogen of this disclosure can be activated into chymotrypsin at activation times of less than 20 hours, 18 hours, 16 hours, 14 hours, 12 hours, 10 hours, 9 hours, 8 hours, 7 hours, 6 hours, 5 hours, 4 hours, or 3 hours. In one embodiment, the recombinant human chymotrypsinogen of this disclosure, after activation into chymotrypsin, has a specific activity substantially the same as or higher than that of naturally extracted human chymotrypsin. In one embodiment, the amino acid sequence of the recombinant human chymotrypsinogen of this disclosure includes the amino acid motif EAEA at its N-terminus. In another embodiment, the amino acid sequence of the recombinant human chymotrypsinogen of this disclosure is obtained by adding EAEA to the N-terminus of the amino acid sequence of a naturally extracted human chymotrypsinogen. In yet another embodiment, the amino acid sequence of the recombinant human chymotrypsinogen of this disclosure is obtained by adding the amino acid motif EAEA to the N-terminus of SEQ ID NO:1.

[0105] In one embodiment, the amino acid sequence of the recombinant human chymotrypsinogen disclosed herein comprises or consists of the following amino acid sequences: (a) the amino acid sequence shown in SEQ ID NO:2; or (b) an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% homology with SEQ ID NO:2; or (c) an amino acid sequence having one or more amino acid substitutions, additions, and / or deletions compared to SEQ ID NO:2.

[0106] In one embodiment, the recombinant human chymotrypsin of this disclosure is derived from human (Homo sapiens).

[0107] V. Nucleotide sequence encoding the recombinant human chymotrypsinogen disclosed herein

[0108] This disclosure provides a nucleotide sequence encoding the recombinant human chymotrypsinogen of this disclosure. In one embodiment, the nucleotide sequence is codon-optimized for an expression system. In one embodiment, the nucleotide sequence is codon-optimized for a yeast expression system. In one embodiment, the nucleotide sequence is codon-optimized for a Pichia pastoris expression system.

[0109] In one embodiment, the nucleotide sequence encoding the recombinant human chymotrypsinogen of this disclosure includes the coding sequence for the amino acid motif EAEA at its 5' end. In one embodiment, the nucleotide sequence encoding the recombinant human chymotrypsinogen of this disclosure includes GAGGCTGAAGCT (SEQ ID NO:10) at its 5' end. In one embodiment, the amino acid sequence of the recombinant human chymotrypsinogen of this disclosure is obtained by adding GAGGCTGAAGCT (SEQ ID NO:10) (SEQ ID NO:10) to the 5' end of the coding sequence of naturally extracted human chymotrypsinogen (e.g., SEQ ID NO:3).

[0110] In one embodiment, the nucleotide sequence of this disclosure comprises or consists of the following nucleotide sequences: (a) the nucleotide sequence shown in SEQ ID NO:4; or (b) a nucleotide sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% homology with SEQ ID NO:4; or (c) a nucleotide sequence having one or more nucleotide substitutions, additions, and / or deletions compared to SEQ ID NO:4.

[0111] VI. The nucleic acid constructs, recombinant expression vectors, and recombinant host cells disclosed herein

[0112] This disclosure provides a nucleic acid construct comprising the nucleotide sequence of this disclosure.

[0113] In one embodiment, the nucleic acid construct or expression vector of this disclosure is obtained by operatively linking the nucleotide sequence of this disclosure to one or more regulatory sequences(s). In one embodiment, the regulatory sequence directs the expression of the coding sequence in a suitable host cell under conditions compatible with the regulatory sequence.

[0114] The regulatory sequence may be a promoter sequence, which is a nucleotide sequence recognized by a host cell for expressing a polynucleotide encoding a polypeptide of the present disclosure. The promoter sequence contains a transcriptional regulatory sequence mediating the expression of the polypeptide. The promoter may be any nucleotide sequence that exhibits transcriptional activity in a selected host cell, including mutant, truncated, and heterozygous promoters, and may be obtained from a gene encoding an extracellular or intracellular polypeptide that is homologous or heterologous to that of the host cell.

[0115] The regulatory sequence can also be a suitable transcription terminator sequence, i.e., a sequence recognized by the host cell to terminate transcription. The terminator sequence is operatively linked to the 3' end of the nucleotide sequence encoding the polypeptide. Any terminator that is functional in a selected host cell can be used in this disclosure.

[0116] The regulatory sequence can also be a polyadenylated sequence, which is a sequence operatively linked to the 3' end of a nucleotide sequence and, during transcription, is recognized by the host cell as a signal to add polyadenylated residues to the transcribed mRNA. Any polyadenylated sequence that is functional in a selected host cell can be used in this disclosure.

[0117] The regulatory sequence can also be a signal peptide-coding sequence that encodes a signal peptide linked to the amino terminus of a polypeptide and directs the encoded polypeptide into the cellular secretion pathway. The 5' end of the coding sequence of the nucleotide sequence may inherently contain a signal peptide-coding sequence, which is naturally linked together with the coding sequence fragment encoding the secreted polypeptide in the translation reading frame. Alternatively, the 5' end of the coding sequence may contain a signal peptide-coding sequence exogenous to said coding sequence. The exogenous signal peptide-coding sequence may be necessary when the coding sequence does not naturally contain a signal peptide-coding sequence. Alternatively, the exogenous signal peptide-coding sequence may simply replace the native signal peptide-coding sequence to enhance polypeptide secretion. However, any signal peptide-coding sequence that directs the expressed polypeptide into the secretion pathway (i.e., secretion into the culture medium) of the selected host cell may be used in this disclosure.

[0118] The regulatory sequence can also be a propeptide-coding sequence, which encodes a propeptide located at the amino terminus of the polypeptide. The resulting polypeptide is called a proenzyme or propolypeptide (or, in some cases, a zymogen). The propeptide is usually inactive and can be converted into a mature, active polypeptide through catalytic or autocatalytic cleavage.

[0119] When both the signal peptide and the propeptide sequence are present at the amino terminus of the polypeptide, the propeptide sequence is placed immediately next to the amino terminus of the polypeptide, and the signal peptide sequence is placed immediately next to the amino terminus of the propeptide sequence.

[0120] Similarly, the addition of regulatory sequences allows for the regulation of polypeptide expression relative to the growth of the host cell. Examples of regulatory systems are those that cause gene expression to turn on or off in response to chemical or physical stimuli, including the presence of regulatory compounds. Regulatory systems in prokaryotes include the lac, tac, and trp gene-operating systems. In yeast, the AOX1 promoter, GAP promoter, FLD1 promoter, TEF promoter, ADH2 system, or GAL1 system can be used. In filamentous fungi, the TAKA α-amylase promoter, the Aspergillus niger glucosylase promoter, and the Aspergillus oryzae glucosylase promoter can be used as regulatory sequences. Other examples of regulatory sequences are those that allow for gene amplification. In eukaryotic systems, these regulatory sequences include dihydrofolate reductase genes amplified in the presence of methotrexate and metallothionein genes amplified with heavy metals. In these cases, the nucleotide sequence encoding the polypeptide will be operatively linked to the regulatory sequence.

[0121] This disclosure provides a recombinant expression vector comprising the nucleotide sequence or nucleic acid construct of this disclosure.

[0122] In one embodiment, the recombinant expression vector of this disclosure comprises the nucleotide sequence of this disclosure, a promoter, and transcription and translation termination signals. Various nucleic acid and regulatory sequences described herein can be combined to produce a recombinant expression vector, which may include one or more convenient restriction sites to allow insertion or substitution of nucleotide sequences encoding polypeptides at these sites. Alternatively, the polynucleotide sequence of this disclosure can be expressed by inserting a nucleotide sequence or nucleic acid construct containing said sequence into a suitable vector for expressing the coding sequence. During the preparation of the expression vector, the coding sequence is placed in the vector, thereby operatively linking the coding sequence to a suitable expression regulatory sequence.

[0123] The recombinant expression vector disclosed herein can be any vector (e.g., plasmid or virus) that facilitates the recombinant DNA step and is capable of producing the expression of a nucleotide sequence. The choice of vector will generally depend on the compatibility of the vector with the host cell to which it will be introduced. The vector can be a linear or closed circular plasmid.

[0124] The recombinant expression vectors disclosed herein can be self-replicating vectors, i.e., vectors that exist as extrachromosomal entities whose replication is independent of chromosome replication, such as plasmids, extrachromosomal elements, minichromosomes, or artificial chromosomes. The vector can contain any means to ensure self-replication. Alternatively, the vector can be one that integrates into the genome when introduced into a host cell and replicates along with the chromosome in which the vector is integrated. Furthermore, single vectors or plasmids, or two or more vectors or plasmids collectively containing the complete DNA of the host cell genome to be introduced, or transposons can be used.

[0125] The recombinant expression vectors disclosed herein preferably contain elements that allow the vector to integrate into the host cell genome or allow the vector to replicate autonomously in the cell independently of the genome.

[0126] To integrate into the host cell genome, the recombinant expression vector of this disclosure may rely on a polynucleotide sequence encoding a polypeptide or any other vector element for integration into the genome via homologous or non-homologous recombination. Alternatively, the vector may contain additional nucleotide sequences to guide integration into the precise location within the host cell genome chromosome via homologous recombination. To increase the likelihood of integration at a precise location, the integrative element preferably contains a sufficient number of nucleic acids that have high sequence identity with the corresponding target sequence to enhance the probability of homologous recombination. The integrative element can be any sequence homologous to the target sequence in the host cell genome. Furthermore, the integrative element can be a non-coding or coding nucleotide sequence. On the other hand, the vector can be integrated into the host cell genome via non-homologous recombination.

[0127] For autonomous replication, the recombinant expression vectors of this disclosure may further include an origin of replication, which enables the vector to replicate autonomously within the host cell. The origin of replication can be any plasmid replicator that mediates autonomous replication and functions within the cell. The terms "origin of replication" or "plasmid replicator" are defined herein as a nucleotide sequence capable of enabling replication within a plasmid or vector.

[0128] Nucleic acid constructs containing more than one copy of the polynucleotide disclosed herein can be inserted into host cells to increase the production of gene products. The increase in the copy number of the polynucleotide can be achieved by integrating at least one additional copy of the sequence into the host cell genome, or by including an amplifiable selective marker gene in the polynucleotide, wherein cells containing an amplified copy of the selective marker gene can be selected by culturing cells in the presence of a suitable selectable agent, thereby selecting cells containing an additional copy of the polynucleotide.

[0129] The methods for connecting the above-described elements to construct the recombinant expression vector of this disclosure are well known to those skilled in the art.

[0130] This disclosure provides a recombinant host cell comprising the nucleic acid construct or expression vector of this disclosure. In one embodiment, the recombinant host cell is a prokaryotic or eukaryotic cell. In one embodiment, the recombinant host cell is a bacterial cell, fungal cell, or animal cell. In one embodiment, the recombinant host cell is an *Escherichia coli* cell. In one embodiment, the recombinant host cell is a *Pichia pastoris* cell.

[0131] In one embodiment, the recombinant host cell of this disclosure comprises the nucleotide sequence of this disclosure, operably linked to one or more regulatory sequences. A construct or vector comprising the nucleotide sequence of this disclosure is introduced into the host cell, such that the construct or vector is maintained as a chromosomal integrase or as a self-replicating extrachromosomal vector as described above. The term "host cell" includes any progeny of the parent cell that differs from the parent cell due to mutations occurring during replication. The selection of the host cell will depend extensively on the gene encoding the polypeptide and its origin.

[0132] In one embodiment, the recombinant host cell of this disclosure may be, for example, a eukaryotic cell, such as a mammalian, insect, plant, or fungal cell.

[0133] VII. Methods of this Disclosure

[0134] This disclosure provides a method for producing the recombinant host cell of this disclosure, comprising: (a) integrating the nucleotide sequence of this disclosure into the genome of a host cell; or (b) converting the nucleic acid construct or expression vector of this disclosure into a host cell.

[0135] In one embodiment, the present disclosure constructs the recombinant host cell of the present disclosure by adding a stop codon TGA to the 3' end of the nucleotide sequence of human chymotrypsinogen (e.g., SEQ ID NO:3) and cloning it into a plasmid vector (e.g., pKJ905M) to obtain a recombinant vector, which is then transformed into a host cell (e.g., Pichia pastoris GS115) to obtain a recombinant host cell that can express the recombinant human chymotrypsinogen of the present disclosure.

[0136] This disclosure provides a method for producing the recombinant human chymotrypsinogen of this disclosure, comprising the following steps: (a) culturing the host cells of this disclosure under conditions favorable for expression of the recombinant human chymotrypsinogen; and (b) recovering the expressed recombinant human chymotrypsinogen from the culture medium. In one embodiment, step (a) comprises: inoculating the recombinant host cells into a shake flask culture medium, culturing them under host-suitable culture conditions (e.g., 30°C, aerated) for a period of time (e.g., 12 hours), then transferring them to a second liquid culture medium (e.g., BSM) at a certain inoculation rate (e.g., 1%–3%), and culturing them again under host-suitable culture conditions (e.g., 30°C, pH 6.0 controlled by supplementing with 25% ammonia, dissolved oxygen value 20%–40%) for a period of time (e.g., 3–12 hours), and after the cell wet weight reaches a certain value, adding methanol for induction, continuing induction culture for a period of time (e.g., 96 hours) before accepting fermentation. In one embodiment, step (b) comprises: purifying the culture from step (a) using chromatography (e.g., cation chromatography). In one embodiment, step (b) includes: centrifuging the above fermentation broth at high speed and collecting the supernatant, and then taking a certain amount (e.g., 1 L) of the supernatant for cation chromatography to obtain the purified recombinant human chymotrypsinogen of this disclosure.

[0137] This disclosure provides a method for producing recombinant human chymotrypsin, comprising the following steps: (a) culturing recombinant host cells of this disclosure under conditions favorable for expression of the recombinant human chymotrypsinogen of this disclosure; (b) recovering the expressed recombinant human chymotrypsinogen from the culture medium; and (c) activating the chymotrypsinogen obtained in step (b) with a chymotrypsinogen activator to obtain recombinant human chymotrypsin. In one embodiment, the chymotrypsinogen activator is trypsin.

[0138] Another method for producing recombinant human chymotrypsin is disclosed, comprising preparing human chymotrypsin A chain and human chymotrypsin B chain as described in any one of the first aspects using a gene recombination method, and then assembling the A chain and B chain to obtain recombinant chymotrypsin.

[0139] Optional preparation methods include (a) culturing recombinant host cells that independently express the A and B chains, respectively, under conditions favorable to the expression of the recombinant human chymotrypsin A and B chains; (b) recovering the expressed recombinant human chymotrypsin A and B chains from the culture medium; and (c) assembling the A and B chains into recombinant human chymotrypsin under conditions favorable to the assembly of the recombinant human chymotrypsin A and B chains.

[0140] Optional preparation methods may include (a) synthesizing the recombinant human chymotrypsin A chain by chemical synthesis by culturing recombinant host cells expressing the B chain separately; (b) recovering the expressed recombinant human chymotrypsin B chain from the culture medium; and (c) assembling the A chain and B chain into recombinant human chymotrypsin under conditions favorable to the assembly of the recombinant human chymotrypsin A chain and B chain.

[0141] IX. Compositions or kits disclosed herein

[0142] A ninth aspect of this disclosure provides a composition or kit comprising the recombinant human chymotrypsinogen of this disclosure; and optionally a pharmaceutically acceptable carrier or excipient.

[0143] In one embodiment, the compositions and kits of this disclosure further comprise a chymotrypsinogen activator. In one embodiment, the chymotrypsinogen activator is trypsin.

[0144] X. Beneficial effects of this disclosure

[0145] The beneficial effects of this disclosure are at least as follows: the recombinant human chymotrypsinogen fused with the EAEA protein tag can be activated into chymotrypsin in approximately 1 / 8 of the activation time of naturally extracted human chymotrypsinogen, significantly shortening the activation time. Furthermore, the recombinant human chymotrypsinogen of this disclosure, after activation into chymotrypsin, exhibits a specific activity substantially the same as or higher than that of naturally extracted human chymotrypsin. Therefore, this disclosure greatly improves the activation efficiency of recombinant human chymotrypsinogen, significantly shortens the production cycle of recombinant human chymotrypsin, and saves production costs. Detailed Implementation

[0146] The present disclosure is further illustrated below by way of embodiments, but these embodiments are not intended to limit the scope of the disclosure. In the following embodiments, unless specific techniques or conditions are specified, they are performed according to the techniques or conditions described in the literature in the art, or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased through legitimate channels.

[0147] Example 1. Construction of human chymotrypsinogen expression strain and identification of expression product

[0148] The amino acid sequence of naturally extracted human chymotrypsinogen hCTRB is shown in SEQ ID NO:1. The amino acid sequence of recombinant human chymotrypsinogen EAEA-hCTRB containing EAEA at the N-terminus is shown in SEQ ID NO:2. The corresponding nucleotide sequences of the two human chymotrypsin amino acid sequences were obtained after codon optimization for the Pichia pastoris expression system, as shown in SEQ ID NO:3 and 4.

[0149] Table 1. Sequences of two human chymotrypsinogens

[0150] Two human chymotrypsinogen nucleotide sequences were cloned into the pKJ905M vector after the 3' end of the sequence was added with the stop codon TGA, thus constructing two recombinant human chymotrypsinogen expression vectors. These vectors were then transformed into Pichia pastoris GS115, and positive recombinant expression strains containing the human chymotrypsinogen nucleotide sequence were obtained by MD plate screening.

[0151] Specifically, five single clones from each of the two human chymotrypsinogen expression strains were inoculated into 5 ml of BMGY in 24-well deep-well plates and cultured at 28°C for 24 hours until the OD600nm reached 10–20. After centrifugation, the bacterial pellet was resuspended in 500 μL of BMGY until the OD600nm reached 100. The pellet was then transferred back into 24-well deep-well plates and cultured at 28°C for 6–8 hours. Methanol was added to a final concentration of 4%, and the plates were cultured for another 16–18 hours. The supernatant was collected for SDS-PAGE electrophoresis.

[0152] The electrophoresis results are shown in Figure 1. The expression level of EAEA-hCTRB is close to that of hCTRB, while the molecular weight of the EAEA-hCTRB expression product is slightly higher than that of hCTRB. N-terminal sequencing of the expression products of EAEA-hCTRB clone 2 and hCTRB clone 2 confirmed that the N-terminal 15 amino acids were completely consistent with the theoretical values, as shown in Table 2.

[0153] Table 2. N-terminal sequencing results

[0154] Example 2. Scale-up fermentation culture of recombinant human chymotrypsinogen

[0155] In Example 1, EAEA-hCTRB clone 2 and hCTRB clone 2 were gradually scaled up to a 5L fermenter for fermentation production via shake flask culture. BSM medium was used as the fermentation medium, employing a fed-batch and submerged aeration method. Glycerol was selected as the pre-induction limiting carbon source for controlled feeding, and methanol was selected as the inducer and post-induction limiting carbon source for controlled feeding. The fermentation temperature was 30℃, and the pH was controlled to 6.0 by adding 25% ammonia. The fermenter aeration rate was selected to be 0.5–2.0 vvm, and the dissolved oxygen level was maintained at approximately 30% by controlling the feeding rate, stirring rate, and aeration rate. During fermentation, samples were taken periodically to determine the wet weight of the cells. Once the wet weight reached a certain value, methanol was added for induction. Fermentation was terminated after 96 hours of induction culture, and the supernatant was collected by centrifugation. 1L of the supernatant sample was purified by cation exchange chromatography to obtain pure chymotrypsinogen protein.

[0156] Example 3. Activation of recombinant human chymotrypsinogen

[0157] The two purified chymotrypsinogen proteins obtained in Example 2 were activated using trypsin. Trypsin and purified chymotrypsinogen proteins were mixed at a ratio of 1:10000 and activated at 2–8°C under pH 7.8 conditions. The activity of the chymotrypsin proteins after different activation times was determined according to the chymotrypsin potency assay method in the 2020 edition of the Chinese Pharmacopoeia.

[0158] As shown in Figure 2, hCTRB requires 24 hours to be fully activated, with a final specific activity of 2027 U / mg. EAEA-hCTRB, on the other hand, requires only 3 hours to be fully activated, with a final specific activity of 2126 U / mg. The final activated activities of the two chymotrypsinogens are essentially the same, but the activation time of EAEA-hCTRB is 21 hours shorter than that of hCTRB.

[0159] Example 4. Analysis of recombinant human chymotrypsin

[0160] The N-terminal sequences of the enzymes activated by hCTRB and EAEA-hCTRB were obtained by Edman degradation method, and two N-terminal sequences were obtained for each enzyme. It was determined that the enzymes activated by both chymotrypsinogens are composed of two peptide chains (A chain and B chain). The N-terminal sequences are shown in Table 3.

[0161] Table 3. Sequencing results of the N-terminus of the activated enzyme

[0162] The enzymes activated by hCTRB and EAEA-hCTRB were reduced using TCEP, and the molecular weight of the reduced proteins was detected by high-resolution mass spectrometry. The detection results are shown in Figures 3 and 4, and the molecular weights are summarized in Table 4.

[0163] Table 4. Molecular weight of enzyme reduction after activation

[0164] Based on the N-terminal sequence and reduced molecular weight results, it was determined that both hCTRB and EAEA-hCTRB activated enzymes consist of two peptide chains, the sequences of which are shown in Table 5.

[0165] Table 5. Activated enzyme peptide chain sequence

[0166] The applicant declares that this disclosure illustrates the detailed methods of this disclosure through the above embodiments, but this disclosure is not limited to the above detailed methods, that is, it does not mean that this disclosure must rely on the above detailed methods to be implemented. Those skilled in the art should understand that any improvements to this disclosure, equivalent substitutions of the raw materials of the disclosed product, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of this disclosure.

[0167] sequence list

[0168] SEQ ID NO:1 Human chymotrypsinogen

[0169] SEQ ID NO:2EAEA-Human Chymotrypsinogen

[0170] SEQ ID NO:3 encodes the nucleotide sequence of human chymotrypsinogen

[0171] SEQ ID NO:4 encodes the nucleotide sequence of EAEA-human chymotrypsinogen

[0172] SEQ ID NO:5 (A chain)

[0173] SEQ ID NO:6 (B chain)

[0174] SEQ ID NO:7 (EAEA-A chain)

[0175] SEQ ID NO:8 (nucleotide sequence encoding the EAEA-A chain)

[0176] SEQ ID NO:9 (nucleotide sequence encoding the B chain)

[0177] SEQ ID NO:10 (nucleotide sequence encoding the protein tag EAEA)

[0178] SEQ ID NO:11 (N-terminal sequencing result of the A strand of EAEA-hCTRB clone 2)

[0179] SEQ ID NO:12 (N-terminal sequencing result of the B chain of the enzyme activated by hCTRB or EAEA-hCTRB)

Claims

1. A human-derived chymotrypsin, wherein, The chymotrypsin is composed of an A chain and a B chain linked by disulfide bonds; the amino acid sequence of the A chain has at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO:5; The amino acid sequence of the B chain has at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO:

6.

2. The human-derived chymotrypsin of claim 1, wherein, The A chain comprises the amino acid sequence shown in SEQ ID NO:5; the B chain comprises the amino acid sequence shown in SEQ ID NO:

6.

3. The human-derived chymotrypsin of claim 1, wherein, The amino acid sequence of chain A is shown in SEQ ID NO:5 or SEQ ID NO:7; the amino acid sequence of chain B is shown in SEQ ID NO:

6.

4. The human-derived chymotrypsin of claim 3, wherein, Chain B is connected to chain A via disulfide bonds formed by C27, C43, C107, C121, C153, C167, C176, C186 or C205; optionally, chain B is connected to chain A via disulfide bonds formed by C107.

5. The human-derived chymotrypsin according to claim 3 or 4, wherein, The B chain also contains at least one intramolecular disulfide bond selected from C27-C43, C121-C186, C153-C167, or C176-C205; Optionally, the B chain also contains intramolecular disulfide bonds C27-C43, C121-C186, C153-C167 and C176-C205.

6. The human-derived chymotrypsin according to any one of claims 1 to 5, wherein, The specific activity of the human chymotrypsin is greater than 1000 units / mg.

7. The human chymotrypsin according to any one of claims 1 to 6, wherein the human chymotrypsin is a recombinant human chymotrypsin.

8. The use of the protein tag EAEA in (a) or (b): (a) Improves the activation efficiency of chymotrypsinogen; (b) Fusing with chymotrypsinogen to prepare recombinant chymotrypsinogen containing the protein tag EAEA, wherein the recombinant chymotrypsinogen containing the protein tag EAEA has a higher activation efficiency than chymotrypsinogen without the protein tag EAEA. Optionally, the protein tag EAEA is located at the N-terminus of the recombinant chymotrypsinogen.

9. The use of claim 8, wherein, The activation time of the recombinant chymotrypsinogen containing the protein tag EAEA is reduced to less than 80%, 70%, 60%, 50%, 40%, 30%, or 20% of the activation time of the chymotrypsinogen without the protein tag EAEA. Optionally, the chymotrypsin obtained by activating the recombinant chymotrypsinogen containing the protein tag EAEA has substantially the same or higher specific activity compared to the chymotrypsin obtained by activating the chymotrypsinogen without the protein tag EAEA.

10. The use according to claim 8 or 9, wherein, The chymotrypsinogen is a human-derived chymotrypsinogen.

11. Use according to any one of claims 8 to 10, wherein, The amino acid sequence of the chymotrypsinogen has at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO:

1.

12. Recombinant chymotrypsinogen with the protein tag EAEA attached to its N-terminus.

13. The recombinant chymotrypsinogen of claim 12, wherein, Compared to chymotrypsinogen without the protein tag EAEA, the activation time of the recombinant chymotrypsinogen is reduced to below 80%, below 70%, below 60%, below 50%, below 40%, below 30%, or below 20%. Optionally, the chymotrypsin obtained by activating the recombinant chymotrypsinogen has substantially the same or higher specific activity compared to the chymotrypsin obtained by activating chymotrypsinogen without the protein tag EAEA.

14. The recombinant prochymase of claim 12 or 13, wherein, The chymotrypsinogen is a human-derived chymotrypsinogen.

15. The recombinant chymotrypsinogen according to any one of claims 12 to 14, wherein the amino acid sequence of the recombinant chymotrypsinogen has at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO:

2.

16. A nucleic acid molecule encoding the recombinant chymotrypsinogen according to any one of claims 12 to 15; Optionally, the nucleotide sequence encoding the protein tag EAEA is GAGGCTGAAGCT (SEQ ID NO:10).

17. A recombinant expression vector comprising the nucleotide sequence of the nucleic acid molecule of claim 16.

18. A recombinant host cell comprising the nucleic acid molecule of claim 16 or the recombinant expression vector of claim 17; Optionally, the host cell is selected from bacterial or fungal cells, such as Escherichia coli cells or Pichia pastoris cells.

19. A method for preparing recombinant chymotrypsinogen according to any one of claims 12 to 15, comprising the following steps: (a) The recombinant host cells of claim 18 are cultured under conditions that favor the expression of the recombinant chymotrypsinogen of any one of claims 12 to 15; (b) Recovery of the expressed recombinant chymotrypsinogen from the culture medium.

20. A method for preparing recombinant chymotrypsin, comprising activating the recombinant chymotrypsinogen according to any one of claims 12 to 15 or the recombinant chymotrypsinogen prepared by the method according to claim 19 with a chymotrypsinogen activator to obtain recombinant chymotrypsin; Optionally, the chymotrypsinogen activator is trypsin.

21. A method for preparing recombinant chymotrypsin, comprising preparing human chymotrypsin A chain and human chymotrypsin B chain as described in any one of claims 1 to 7, and then assembling the A chain and B chain to obtain recombinant chymotrypsin. Optionally, the A chain can be synthesized by chemical methods, and the B chain can be prepared by gene recombination methods.

22. A pharmaceutical composition comprising (a) a therapeutically effective amount of the human chymotrypsin according to any one of claims 1 to 7 or a recombinant chymotrypsin prepared by the method of preparing the recombinant chymotrypsin according to claim 20 or 21, and (b) a pharmaceutically acceptable carrier.

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