CN115386565A - Human chymotrypsinogen mutant and application thereof - Google Patents

Abstract

The invention discloses a human chymotrypsinogen mutant and application thereof. The amino acid sequence of the human chymotrypsinogen is shown in SEQ ID NO. 2; replacing the 114 th site of the human chymotrypsinogen to obtain the mutant; the substitution at position 114 with alanine, arginine, asparagine, glutamic acid, glutamine, histidine, lysine, proline, serine, threonine, or valine; the function of the mutant comprises at least the function of chymotrypsin. The human chymotrypsinogen mutant provided by the invention has high heat stability of the chymotrypsin prepared by utilizing the human chymotrypsinogen mutant, and is more suitable for developing and preparing medicines for treating and preventing surgical inflammation, trauma, hematoma and abscess, postoperative inflammation and organ inflammation diseases or symptoms and applying to combined use with other therapeutic agents.

Description

Human chymotrypsinogen mutant and application thereof

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a human chymotrypsinogen mutant and application thereof.

Background

Chymotrypsin (EC 3.4.21.1), also called chymotrypsin, belongs to serine protease family, is produced in vivo in zymogen form, forms active chymotrypsin through trypsin and autohydrolysis, and mainly hydrolyzes carboxyl-terminal peptide chain with large-scale hydrophobic amino acid residue as side chain, such as Tyr, trp, phe and Leu.

The chymotrypsin is widely applied clinically and is mainly used for inflammation, inflammatory edema, hematoma, adhesion, ulcer and the like after surgical operation or trauma; chest abscess, bloody chest, difficult expectoration after operation, lung flatulence, etc.; medical chronic bronchitis, bronchial asthma, etc.; cervicitis, salpingitis, pelvic inflammation and the like in obstetrics and gynecology department; treating keratitis and suppurative otitis media in ophthalmology and ophthalmology.

At present, chymotrypsin is mainly extracted from pancreas of pigs and cattle, and has risks of unknown virus and exogenous factor pollution due to animal origin, so that the application of the chymotrypsin in the pharmaceutical industry is greatly limited; in addition, due to the similar structure and properties of trypsin and chymotrypsin, complete single chymotrypsin cannot be obtained through extraction, separation and purification of raw materials, and even high-purity chymotrypsin is difficult to obtain. The chymotrypsinogen is expressed by the recombinant engineering bacteria, and the chymotrypsin with high purity and high activity can be produced. The non-animal source recombinant human chymotrypsin replaces other chymotrypsin extracted from animal pancreas, which meets the development trend of biological medicines, and the field needs to produce the human source chymotrypsin in large scale. However, the natural human chymotrypsin has poor stability, and the popularization and the application of the natural human chymotrypsin are greatly limited. Therefore, there is a need in the art to improve the stability of human chymotrypsin.

Balakrishnan et al report that the substrate specificity of ChymotrypsiN mutants derived from human (V99M/M192R/G218R/V219E/S221T/T222G/S223T/T224W/A226G) is expanded, and the half-life is improved by 8 times compared with that of WT, but because of too many mutation sites, immunogenicity problems are easily caused, and the human is at a high risk of drug administration (Ramesh B, abnouf S, mali S, et al. Considering that amino acid changes are easy to bring immunogenicity, mutation transformation is performed as little as possible during sequence design, so that mutants with less amino acid transformation and improved stability need to be designed and screened.

Disclosure of Invention

The invention aims to solve the technical problem of overcoming the defect that the prior art is lack of human chymotrypsin with high stability and provides a human chymotrypsinogen mutant and application thereof. Compared with a wild type, the thermal stability of the human chymotrypsin obtained by utilizing the chymotrypsinogen mutant of the invention is obviously improved.

1. Mutants

The invention provides a mutant of human chymotrypsinogen, which comprises an amino acid sequence shown in a sequence table SEQ ID NO. 2; the mutant is obtained after replacing the position 114 of the amino acid sequence, and the 114 th position is replaced by alanine, arginine, asparagine, glutamic acid, glutamine, histidine, lysine, proline, serine, threonine or valine; the function of the mutant at least comprises the function of the chymotrypsinogen.

By "comprising at least" the function of chymotrypsinogen is meant that the mutant may also comprise other functions on the basis of retaining the function of chymotrypsinogen.

The mutant is preferably produced by a genetic engineering recombination mode.

Preferably, the amino acid at position 114 is substituted, and the phenylalanine (F) is substituted for any other amino acid at position 114 of the mutant corresponding to SEQ ID NO: 2.

A substitution of phenylalanine (F) for alanine (A), arginine (R), asparagine (N), glutamic acid (E), glutamine (Q), histidine (H), lysine (K), proline (P), serine (S), or valine (V) or threonine (T) at position 114 of the mutant corresponding to human chymotrypsinogen (SEQ ID NO: 2); namely F114A, F114R, F114N, F114E, F114Q, F114H, F114K, F114P, F114S, F114V or F114T.

More preferably, the amino acid phenylalanine at position 114 is replaced with any one of proline (P) or threonine (T); i.e., F114P or F114T.

In the invention, the amino acid sequence of the human chymotrypsinogen is preferably shown in any one sequence of SEQ ID NO 3-8 and 10-14.

Preferably, the mutant of the present invention may be further mutated, and the sequence of the mutant obtained by the further mutation has a sequence homology of 80% or more, 85% or more, 90% or more, 95% or more, or 99% or more with SEQ ID No. 2, and at least includes the function of chymotrypsinogen. The wild type human chymotrypsinogen amino acid sequence related by the invention is as follows:

<xnotran> CGVPAIHPVLSGLSRIVNGEDAVPGSWPWQVSLQDKTGFHFCGGSLISEDWVVTAAHCGVRTSDVVVAGEFDQGSDEENIQVLKIAKVFKNPKFSILTVNNDITLLKLATPARFSQTVSAVCLPSADDDFPAGTLCATTGWGKTKYNANKTPDKLQQAALPLLSNAECKKSWGRRITDVMICAGASGVSSCMGDSGGPLVCQKDGAWTLVGIVSWGSDTCSTSSPGVYARVTKLIPWVQKILAAN (SEQ ID NO: 2) . </xnotran>

Synthesizing corresponding nucleotide sequences after codon optimization preferred by pichia pastoris, respectively cloning to expression vectors, converting pichia pastoris, carrying out induction culture on the converted yeast, separating a human chymotrypsinogen mutant from culture supernatant through nickel metal affinity chromatography, and finally activating the human chymotrypsinogen enzyme digestion through trypsin to obtain the chymotrypsinogen mutant.

The invention is characterized in that the 114 th amino acid of the human chymotrypsinogen is mutated into Ala, arg, asn, glu, gln, his, lys, pro, ser, thr or Val from Phe, and the chymotrypsin heat stability and the freeze-thaw stability obtained by utilizing the mutated human chymotrypsinogen are obviously improved.

The complete chymotrypsin sequence used in the present invention is publicly available as UniProt sequence No. P17538, which comprises a secretion signal peptide sequence of 18 amino acids. This sequence is typically removed to form mature chymotrypsinogen, the sequence of which is provided herein as SEQ ID NO: 2. All references to numbering of amino acid positions in the immunoglobulin degrading zymogen sequences disclosed herein are based on the numbering of the corresponding positions in SEQ ID NO 2 from the N-terminus, unless otherwise indicated.

The present invention also provides a secreted protein comprising a mutant as described above.

Preferably, the secreted protein has a purification tag such as GST tag, MBP tag, SUMO tag, FLAG tag, C-Myc tag, or histidine tag at the N-terminus and/or C-terminus. The sequence of the histidine tag is preferably HHHHHHHHHH or HHHHHHHHHHHHHHHH.

The invention also provides a nucleotide for coding the human chymotrypsinogen.

The invention also provides an expression vector containing the nucleotide; preferably, the expression vector further comprises a nucleotide encoding a signal peptide prior to the nucleotide; the amino acid sequence of the signal peptide is preferably shown as MVAWWSLFLYGLQVAAPALA (SEQ ID NO: 1).

The present invention also provides a host cell comprising an expression vector as described above, or a host cell expressing a mutant of human chymotrypsinogen as described above.

The host cell may be a cell conventionally used in the art for expressing a protein or polypeptide, preferably a yeast cell or an E.coli cell.

The invention also provides the human chymotrypsin prepared from the human chymotrypsinogen mutant.

According to the common knowledge in the art, human chymotrypsin comprises 3 chains, and the amino acid sequences of the 3 chains of human chymotrypsin in the present invention are preferably shown in SEQ ID NO. 15-17, respectively.

A method of preparing human chymotrypsin as described above comprising: a mutant that activates human chymotrypsinogen as described above; preferably, the activation is performed using trypsin;

preferably, the method further comprises: culturing said host cell to obtain said mutant of human chymotrypsinogen prior to said activation.

2. Use of mutants

2.1 degradation of proteins

The invention also provides the application of the human chymotrypsinogen mutant or the human chymotrypsin in protein degradation. The application is, for example, mass spectrum molecular weight analysis, glycoform modification analysis, peptide spectrogram in proteomics research, peptide fingerprint spectrum or protein sequence analysis and the like.

2.2 preparation of the medicament

The invention also provides the application of the human chymotrypsinogen mutant or the human chymotrypsin in preparing medicaments for treating diseases. Preferably, the medicament further comprises a pharmaceutically acceptable carrier or excipient.

Preferably, wherein the variant of human chymotrypsin or human chymotrypsin as described above is administered by intramuscular injection, infusion, spraying and topical application. The specific dosage and mode of administration will be determined by the clinician.

Preferably, the disease is surgical inflammation, trauma, hematoma, abscess, etc., and also can be used for postoperative inflammation, organ inflammation diseases or symptoms; preferably, the disease is inflammation, inflammatory edema, hematoma, adhesion, ulcer, empyema, hemothorax, pleurisy, postoperative expectoration difficulty, pneumonia, bronchitis, pharyngitis, laryngitis, rhinitis, bronchial asthma, chronic Obstructive Pulmonary Disease (COPD), respiratory tract burn, nasosinusitis, otitis media, pharyngolaryngitis, tonsillitis, herpetic stomatitis, keratitis, acute oropharyngeal tube obstruction, vocal cord polyp, vocal nodule operation, tracheotomy, nasal endoscopic sinus surgery, cervicitis, salpingitis, pelvic inflammatory disease, intracranial hematoma, brain abscess, perianal abscess, periodontal abscess, various deep abscesses such as thymic abscess, body surface cyst, ganglion cyst, skin chronic ulcer, bedsore, acne, venomous snake bite, diabetic foot ulcer, operation wound fatty liquefaction and the like after surgical operation or trauma.

3. Composition comprising a metal oxide and a metal oxide

The invention also provides a composition, in particular a pharmaceutical composition or a pharmaceutical combination, comprising a mutant of human chymotrypsinogen or human chymotrypsin as described above, and a therapeutic agent; when the composition comprises a mutant of human chymotrypsinogen, the composition further comprises an enzyme for activating the mutant of human chymotrypsinogen. Wherein the medicine can be expectorant, antibiotic, hormone, other Chinese medicinal materials with debridement, antiinflammatory and antibacterial effects, enzymes, growth factor, etc.; preferably, the composition further comprises a pharmaceutically acceptable carrier or excipient.

Preferably, the composition as described above may comprise a expectorant. The expectorant comprises mucolytic agent, standard Myrtus communis oil, etc. The mucolytic agent is preferably ambroxol hydrochloride. The ambroxol hydrochloride can increase the secretion of respiratory tract mucosa serous gland and reduce the secretion of mucous gland, thereby reducing the viscosity of sputum. Standard myrtle oil can reestablish the clearance function of mucociliary clearance systems of upper and lower respiratory tracts, thereby thinning and alkalizing mucus, enhancing mucociliary movement, obviously increasing the mucus moving speed and promoting sputum excretion.

Preferably, the composition as described above may comprise an antibiotic drug, such as one or more of amikacin sulphate, amoxicillin clavulanate potassium, metronidazole, triamcinolone acetonide, gentamicin and tobramycin.

Preferably, the composition as described above may comprise a hormonal drug, prednisolone, triamcinolone acetonide, fluorometholone.

Preferably, the composition can contain other traditional Chinese medicines, enzymes, growth factors and the like with debridement, anti-inflammation and bacteriostasis effects. Such as Saviae Miltiorrhizae radix, yunnan white drug powder, anisodamine, stannum powder, behcet, FUKANG cream, KANGFUXIN, trypsin, sodium hyaluronate, and vitamin B6.

In particular, for the pharmaceutical combination as described above, it may further comprise ambroxol hydrochloride, dexamethasone acetate, dexamethasone sodium phosphate, standard myrtle oil, salvia miltiorrhiza, yunnan white drug powder, anisodamine, tobramycin, prednisolone acetate, methylprednisolone sodium succinate, amikacin sulfate, amoxicillin potassium clavulanate, metronidazole, triamcinolone acetonide, gentamycin, flumethasone, budesonide, stannic powder, benexin, fukangfang cream, kangfuxin, trypsin, sodium hyaluronate, vitamin B6, omeprazole, 5% sodium bicarbonate.

Preferably, the pharmaceutical combination further comprises a medical detection reagent, and the drug is selected from antifoaming agents in gastroscopy, local anesthetics such as dimethicone and dyclonine hydrochloride.

4. Use of a composition

The invention also provides the application of the composition in preparing a medicament for treating or preventing diseases. Preferably, the disease is profuse sputum and inflammation of the ear, nose, throat and respiratory tract, traumatic wounds and surgical wounds, surface cysts and ganglions, venomous snake bites, diabetic feet, and the like.

Preferably, the sputum and the inflammations of eyes, ears, throats and respiratory tracts are inflammatory lesions in the respiratory tracts, and the sputum is composed of components such as mucus, foreign matters, pathogenic microorganisms, various inflammatory cells, necrotic and desquamated mucosal epithelial cells and the like. The diseases include chronic dacryocystitis, acute rhinitis, acute pharyngitis, acute laryngitis, acute bronchitis, acute sinusitis, acute Eustachian tube obstruction, chronic pharyngitis vocal cord polyp, vesicular stomatitis, vocal nodule operation, tracheotomy operation, nasal endoscopic sinus operation and other upper and lower respiratory tract infections, bronchitis, pneumonia, tuberculosis, asthma, chronic Obstructive Pulmonary Disease (COPD), respiratory tract burn and other respiratory system diseases, and relieve excessive phlegm symptoms caused by other factors.

The trauma wound and the operation wound comprise various trauma inflammations, ulcers, bedsores, periapical inflammation, wound treatment after operation and fat liquefaction of the operation wound; the postoperative wound also comprises anorectal disease operation wound and uterine cavity operation wound; the anorectal disease operation also comprises anal fissure, hemorrhoids, anal abscess, anal fistula, anal sinusitis and the like.

The internal inflammation comprises postoperative pelvic cavity and abdominal cavity adhesion, ulcerative colitis, osteoarticular tuberculosis combined abscess, gastrolithiasis and the like.

The body surface cyst and ganglion cyst diseases include ganglion cyst, cystic acne, sebaceous cyst, epidermoid cyst, dermatoid cyst, etc.

The diabetic foot is Diabetic Foot Ulcer (DFU), which is characterized in that firstly, slough and necrotic tissues are removed through wound debridement and infection is controlled, and then comprehensive internal and surgical treatment is carried out to accelerate wound healing and repair wound surface.

In the use of the present invention, the human chymotrypsin and therapeutic agent as described above are present as a combined preparation for simultaneous, separate or sequential use.

Preferably, the human chymotrypsin as described above is administered before and/or simultaneously and/or after the administration of the expectorant, antibiotic, hormone, other Chinese drugs with debridement, anti-inflammatory and bacteriostatic effects, enzymes, growth factors, etc.

In the use of the present invention, the human chymotrypsin and therapeutic agent as described above are present as a combined preparation for simultaneous, separate or sequential use.

In some embodiments, the method comprises the steps of: 1) Administering to a subject a human chymotrypsin as described above; subsequently, 2) administering the therapeutic agent to the subject. Preferably, the administration of human chymotrypsin and the therapeutic agent as described above occurs at a time interval.

In some embodiments, the method comprises the steps of: 1) Administering the therapeutic agent to the subject; subsequently, 2) administering to the subject a human chymotrypsin as described above. Preferably, the administration of human chymotrypsin and the therapeutic agent as described above occurs at a time interval.

Preferably wherein the human chymotrypsin as described above is administered by aerosol inhalation, intramuscular injection, infusion and topical application. Preferably, the administration of thick sputum and inflammation of the ear, nose, throat and respiratory tract is by aerosol inhalation; preferably, the mode of applying the medicine to the trauma wound and the operation wound is local external application; the cyst of body surface and ganglion are injected subcutaneously and injected into joints; the general administration method of the poisonous snake bite is that the mutant medicine is injected at multiple points around the poisonous snake bite to form annular closure, and the injection is infiltrated at the bite part.

Before use, the product is dissolved in a proper amount of sodium chloride injection, and the use method comprises 1) intramuscular injection of 4000 units once; 2) Injecting into posterior chamber, and flushing the left medicine in anterior chamber and posterior chamber with sodium chloride injection after one time of 800 units for 3 minutes; 3) Wet dressing the wound surface, determining the amount of the product according to the size of the wound surface, soaking a sliver after a proper amount of sodium chloride injection is dissolved, and applying the sliver on the wound surface; 4) Atomizing and inhaling, namely mixing 4000 units of physiological saline at a time and then atomizing and inhaling by using an atomizer.

The specific dosage and mode of administration of the proteins of the invention may also be determined by the clinician.

5. Medicine box

The invention also provides a kit or kit of parts for use in the prevention or treatment of an infection, the kit comprising: 1) A therapeutically effective amount of a medicament comprising a human chymotrypsin as described above; and 2) a therapeutically effective amount of a therapeutic agent; the therapeutic agent is selected from expectorants, antibiotics, hormones, other Chinese medicinal materials with debridement, antiinflammatory and antibacterial effects, enzymes and growth factors.

The kit comprises a kit A and a kit B, wherein the kit A comprises a therapeutically effective amount of the human chymotrypsin and/or the human chymotrypsinogen mutant, and the kit B preferably comprises a therapeutically effective amount of a therapeutic agent. The kit preferably further comprises kit C medical detection reagents. When the mutant human chymotrypsinogen is contained in kit a, the kit of parts may further comprise a further kit comprising trypsin for activating the mutant human chymotrypsinogen. The therapeutic agent is selected from expectorant, antibiotic, hormone, other Chinese medicinal materials with debridement, antiinflammatory and antibacterial effects, enzymes and growth factor, and the medical detection reagent is selected from local anesthetic and defoaming agent for gastroscopy. The phlegm-discharging medicine is as follows: ambroxol hydrochloride and standard myrtle oil; antibiotics, such as amikacin sulfate, amoxicillin clavulanate potassium, metronidazole, triamcinolone acetonide, gentamicin, and tobramycin; the other Chinese medicinal materials, enzymes and growth factors with debridement, antiinflammatory and antibacterial effects, such as Saviae Miltiorrhizae radix, yunnan white drug powder, anisodamine, stannum powder, BEIFUXIN, FUKANGSHUANG, KANGFUXIN, trypsin, sodium hyaluronate, and vitamin B6; the defoaming agent is simethicone or dyclonine hydrochloride.

The kit can comprise instructions for administering (e.g., dosage information, dosing interval information) a therapeutically effective amount of a pharmaceutical composition comprising human chymotrypsin as described above and a therapeutically effective amount of a therapeutic agent. The therapeutic agent is selected from expectorants, antibiotics, hormones, other enzymes or growth factors with debridement effect; the preferred expectorant is ambroxol hydrochloride; the antibiotics are preferably amikacin sulfate, amoxicillin potassium clavulanate, metronidazole, triamcinolone acetonide, gentamicin and tobramycin; the hormone is preferably prednisolone acetate, triamcinolone acetonide, and dexamethasone; the other enzymes or growth factors having debridement action are preferably trypsin.

In any aspect of the invention, administration of the human chymase and the therapeutically effective amount of the therapeutic agent as described above may be simultaneous, separate or sequential, e.g., for the treatment and/or prevention of thick sputum and inflammation of the ear, nose, throat and respiratory tract, traumatic wounds, surface and ganglion cysts, venomous snake bites, diabetic foot ulcers and/or surgical wound infections. The mutant or protein and therapeutic agent drug as described above may be provided as separate formulations or as a combined formulation.

The pharmaceutical carrier may be a liquid, and the pharmaceutical composition may be in the form of a solution. Liquid carriers are used in preparing solutions, suspensions, emulsions, syrups, elixirs and pressurized compositions. The active ingredient may be dissolved or suspended in a pharmaceutically acceptable liquid carrier such as water, an organic solvent, a mixture of the two, or a pharmaceutically acceptable oil or fat.

Pharmaceutical compositions for parenteral administration are sterile, substantially isotonic, pyrogen-free and are prepared in accordance with GMP by the FDA or similar agencies. Auxiliary substances, such as wetting or emulsifying agents, surfactants, and pH buffering substances, and the like, may be present in the composition. Other components of the pharmaceutical compositions are those of petroleum, animal, vegetable or synthetic origin, for example peanut oil, soybean oil and mineral oil. Generally, glycols such as propylene glycol or polyethylene glycol are preferred liquid carriers, especially for injectable solutions. Generally, the compositions are prepared as injectables, either as liquid solutions or suspensions; solid forms suitable for dissolution or suspension in a liquid carrier prior to injection may also be prepared.

Unless defined otherwise, 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 invention belongs. Although any methods, devices, and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods, devices, and materials are now described.

The term "nucleotide" or "polynucleotide" means a deoxyribonucleotide, deoxyribonucleoside, ribonucleoside, or ribonucleotide, and polymers thereof, in either single-or double-stranded form. Unless specifically limited, the term encompasses nucleic acids containing known analogs of natural nucleotides that have binding properties similar to the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise specifically limited, the term also means oligonucleotide analogs, which include PNAs (peptide nucleic acids), DNA analogs used in antisense technology (phosphorothioates, phosphoramidates, and the like). Unless otherwise specified, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (including, but not limited to, degenerate codon substitutions) and complementary sequences as well as the sequence explicitly specified. In particular, degenerate codon substitutions may be achieved by generating sequences in which the 3 rd position of one or more selected (or all) codons is substituted with mixed base and/or deoxyinosine residues (Batzer et al, nucleic Acid Res.19:5081 (1991); ohtsuka et al, J.biol.chem.260:2605-2608 (1985); and Cassol et al (1992); rossolini et al, mol cell.Probes8:91-98 (1994)).

The terms "polypeptide" and "protein" are used interchangeably herein to mean a polymer of amino acid residues. That is, the description for a polypeptide applies equally to the description of a peptide and to the description of a protein, and vice versa. The terms apply to naturally occurring amino acid polymers as well as amino acid polymers in which one or more amino acid residues is a non-naturally encoded amino acid. As used herein, the term encompasses amino acid chains of any length, including full-length proteins, in which the amino acid residues are linked via covalent peptide bonds.

The term "host cell" means a cell comprising a nucleotide of the invention, regardless of the method used for insertion to produce a recombinant host cell, e.g., direct uptake, transduction, pairing, or other methods known in the art. The exogenous polynucleotide may remain as a non-integrating vector, such as a plasmid, or may integrate into the host genome. The host cell may be a prokaryotic cell or a eukaryotic cell.

The term "transformation" means the process of introducing a heterologous DNA sequence into a host cell or organism.

The term "expression" means the transcription and/or translation of an endogenous gene or transgene in a cell.

The positive progress effects of the invention are as follows:

the stability of the chymotrypsin obtained by the human chymotrypsinogen mutant is higher than that of wild chymotrypsin, and the chymotrypsin mutant is more suitable for developing and preparing medicines for treating debridement and anti-inflammation and combined use with other therapeutic agents (sputum excretion medicines, antibiotics, hormones, hydrolytic enzymes and other medicines).

Drawings

FIG. 1 is a schematic diagram of electrophoresis of wild-type and mutant protein expression; wherein M is 116kD marker, and 1-13 are F114A, WT, F114T, F114I, F114P, F114R, F114N, F114E, F114Q, F114H, F114K, F114S and F114V, respectively.

FIG. 2 shows the comparison of the activity of wild-type and mutant proteins.

FIG. 3A shows the results of the 25 ℃ stability test of wild-type and mutant proteins; FIG. 3B shows the results of 37 ℃ stability detection of wild-type and mutant proteins; FIG. 3C shows the results of 50 ℃ stability assays for wild-type and mutant proteins.

FIG. 4 shows the results of 5 times of freeze-thawing residual activities of wild-type and mutant proteins.

Detailed Description

The invention is further illustrated by the following examples, which are not intended to limit the scope of the invention. The experimental methods without specifying specific conditions in the following examples were selected according to the conventional methods and conditions, or according to the commercial instructions.

Example 1 expression of mutants

Design of point mutations

After analyzing the properties of other 19 amino acids in which Phe at the 114 th site of a wild-type human chymotrypsinogen sequence (SEQ ID NO: 2) can be mutated, selecting the mutated amino acid as Ala, arg, asn, glu, gln, his, ile, lys, pro, ser, thr or Val, and sequentially obtaining mutants F114A, F114R, F114N, F114E, F114Q, F114H, F114I, F114K, F114P, F114S, F114T and F114V, wherein the mutants are shown in the following table.

TABLE 1

Variant numbering	Modification relative to wild type (SEQ ID NO: 2)
		Variant 1	F114A
Variant
	2	F114R
Variant
	3	F114N
Variant 4			F114E
	Variant
5		F114Q
	Variant
6		F114H
	Variant
7		F114I
	Variant
8		F114K
	Variant
9		F114P
	Variant
10		F114S
	Variant
11		F114T
	Variants
12		F114V

A polynucleotide sequence for coding a mutant is synthesized by the Tokyo Jinsri biotechnologies Limited company through codon optimization preferred by pichia pastoris, an N-terminal alpha-amylase signal peptide sequence MVAWWSLFLYGLQVAAPALA (SEQ ID NO: 1) and a C-terminal 6 Xhistidine tag HHHHHHHHHH are added, the obtained product is inserted into a pPink-HC plasmid after synthesis, and the recombinant plasmid for wild type and mutant expression is obtained after correct sequencing.

Expression of the mutant

The wild type and mutant expression plasmids prepared above were linearized by SpeI and then electrically transformed into pichia pink2 cells (Thermo Fisher, a 11154), coated with a PAD plate, and then placed in a 30 ℃ incubator for inverted culture for 2-3 days. Respectively picking 3 white colonies of the wild type and the mutant, inoculating the white colonies into a 24-hole deep-hole plate in 5ml BMGY, culturing at 28 ℃ for 24h until the OD reaches 10-20, taking out a certain bacterial liquid, centrifuging, and then re-suspending the bacterial precipitate with 500ul BMMY until the OD reaches 100. Then transferring the mixture into a deep-hole plate with 24 holes, culturing for 6-8 h at 28 ℃, adding methanol until the final concentration is 4%, and continuing culturing for 16-18 h. Supernatants were collected for SDS-PAGE detection.

Results

The plasmid containing the coding mutant is transformed into a yeast Pichia Pink2 expression strain for expression and identification, and as shown in figure 1, an obvious expression strip can be seen.

Example 2 comparison of the Activity of mutant chymotrypsin

Expression of mutant human chymotrypsinogen

Selecting a mutant and a wild chymotrypsin expression strain with expression quantity not obviously lower than that of a wild type, inoculating the mutant and the wild type chymotrypsin expression strain into a BMGY culture medium, and culturing at 28 ℃ until OD600 reaches 4-6. After the bacterial liquid is centrifuged, the bacterial cells are resuspended to OD600 about 1 by using BMMY culture medium, and cultured at 28 ℃. Adding 1% methanol every day, culturing for 6-7 days, collecting liquid, centrifuging, and detecting supernatant by SDS-PAGE.

Purification of mutant human chymotrypsinogen

Wild type and mutant expression supernatants were concentrated approximately 5-fold using an ultrafiltration centrifuge tube and exchanged into 20mM PB,500mM NaCl, pH7.4 buffer. And (3) purifying the human chymotrypsinogen by adopting Zhongkensihuida-Ni agarose magnetic beads. 0.5ml of Sepharose beads was treated 2 times with 5 volumes of 20mM Phosphonate buffer,500mM NaCl,20mM Imidazole, pH7.4, and then the concentrated sample was thoroughly mixed with the beads for 30min. The beads were washed with 5 volumes of 20mM Phosphonate buffer,500mM NaCl,20mM Imidazole, pH 7.4. The wash was collected and OD280 was detected, and the wash was repeated until OD280 was less than 0.1. The beads were washed with 2 volumes of 20mM Phosphalate buffer,500mM NaCl,300mM Imidazole, pH7.4 eluent. The eluate was collected and the OD280 detected, and the elution was repeated until the OD280 was less than 0.1.SDS-PAGE detects the protein purity in the eluate. The eluates with purity >90% were mixed and exchanged into phosphate buffer using ultrafiltration centrifuge tubes.

Activation of mutant human chymotrypsinogen

Adopts trypsin to catalyze and activate human chymotrypsinogen. Trypsin is mixed with wild type or mutant human chymotrypsinogen according to the proportion of 1. And directly detecting the activity of the activated chymotrypsin.

Activity detection

The chymotrypsin is detected by adopting the chymotrypsin determination method of 2020 edition of Chinese pharmacopoeia. 0.2ml of 0.0012mol/L hydrochloric acid solution and 3.0ml of substrate solution were taken, and measured at a wavelength of 237nm at 25 ℃ and adjusted to an absorbance of 0.2. Then 0.2ml of the test solution is precisely taken, 3.0ml of the substrate solution is added, timing and shaking are carried out immediately, the absorbance is read every 30 seconds for 5 minutes (repeated once), the change rate of the absorbance is constant, and the constant time is not less than 3 minutes. If the rate of change is not held constant, it can be measured separately at lower concentrations. The rate of change of absorbance per 30 seconds should be controlled to 0.008 to 0.012, the absorbance is plotted as ordinate and the time as abscissa, and the absorbance of the linear portion formed within 3 minutes is calculated by the following formula. The calculation formula is as follows:

in the formula: p is the titer of each lmg of chymotrypsin, and the unit is unit/mg;

a2 is the absorbance starting on the straight line;

a1 is the absorbance ending on a straight line;

t is the time in minutes from A2 to A1 reading;

w is the amount of the test sample in mg in the determination solution;

0.0075 is a change in absorbance per minute of 0.0075 under the above conditions, which corresponds to 1 chymotrypsin unit.

Results

The results are shown in FIG. 2. And obtaining three mutants of F114P, F114I and F114T with activity close to that of the wild type by activity detection and screening.

Example 3 comparison of the thermostability of wild-type and mutant human chymotrypsin

Variation of enzyme activity

The human chymotrypsinogen prepared in example 2 was activated according to the method of example 2. The activated enzyme solution is changed to a buffer solution with the pH value of 7.0, and the activity is measured after the enzyme solution is respectively placed in a water bath with the temperature of 25 ℃, 37 ℃ and 50 ℃ for incubation for 0h, 3h, 6h and 24h. Taking the initial activity of the enzyme solution before water bath as 100 percent, and calculating the residual enzyme activity at the corresponding temperature.

Results

The temperature stability of the wild type and the mutant are shown in FIGS. 3A to 3C, and the change of the enzyme activity of the mutant F114P, F114I, F114T and the wild type WT protein in a buffer solution with pH7.0 is considered within 24h at 25-50 ℃. With the temperature rise, the thermal stability of the enzyme is continuously reduced, the wild type activity is reduced to about 60% after the enzyme is incubated for 24h at 25 ℃ (figure 3A), the activity change of F114T is small, 66% of F114P remains, and 82% of F114I remains; after incubation at 37 ℃ for 24 hours (figure 3B), the residual enzyme activity of the wild type is reduced to 15 percent, the F114T enzyme activity with the best residual activity is remained by 45 percent, and the F114P enzyme activity is remained by 30 percent; when the temperature reaches 50 ℃ (figure 3C), the activity of the wild type is reduced to be basically inactive within 3h, and about 25% of the F114T enzyme activity remains; among F114P, F114I and F114T, F114T has the best thermal stability at 37 ℃, and the heat resistance is better than that of the wild type.

Example 4 comparison of Freeze-thaw stability of wild-type and mutant chymotrypsin

Variation of enzyme activity

The human chymotrypsinogen prepared in example 2 was activated according to the method of example 2. And (3) changing the activated enzyme solution into a buffer solution with the pH value of 7.0, subpackaging a plurality of tubes, placing the tubes in a refrigerator at the temperature of-70 ℃ for freezing, taking out the sample, placing the sample at the room temperature for a period of time until the solution is completely melted, and then placing the sample tubes in the refrigerator for freezing. The samples were freeze-thawed 5 times repeatedly. Taking the initial activity of the enzyme solution before freeze thawing as 100%, and calculating the residual enzyme activity under different freeze thawing times.

Results

The temperature stability of the wild type and the mutant are shown in fig. 4, and the change of the enzyme activities of the mutant F114P, F114I, F114T and the wild type WT protein in 5 times of freeze-thawing is considered. After 5 times of freeze thawing, the activity of the wild type is reduced to about 63 percent, 71 percent of F114I remains, 78 percent of F114P remains, and 85 percent of the F114T enzyme activity with the best residual activity remains; F114P, F114I and F114T are all improved in stability, the residual activity of F114T and F114P is about 20% and 10% higher than that of F114I, and the freeze-thaw performance of the composite is better than that of wild type and F114I.

The applicant states that the present invention is illustrated in detail by the above examples, but the present invention is not limited to the above detailed methods, i.e. it is not meant that the present invention must rely on the above detailed methods for its implementation. It should be understood by those skilled in the art that any modification of the present invention, equivalent substitutions of the raw materials of the product of the present invention, addition of auxiliary components, selection of specific modes, etc., are within the scope and disclosure of the present invention.

SEQUENCE LISTING

<110> Shanghai Baoji pharmaceutical Co., ltd

<120> human chymotrypsinogen mutant and application thereof

<130> P20015739C

<160> 17

<170> PatentIn version 3.5

<210> 1

<211> 20

<212> PRT

<213> Artificial Sequence

<220>

<223> Signal peptide

<400> 1

Met Val Ala Trp Trp Ser Leu Phe Leu Tyr Gly Leu Gln Val Ala Ala

1 5 10 15

Pro Ala Leu Ala

20

<210> 2

<211> 245

<212> PRT

<213> Homo sapiens

<400> 2

Cys Gly Val Pro Ala Ile His Pro Val Leu Ser Gly Leu Ser Arg Ile

1 5 10 15

Val Asn Gly Glu Asp Ala Val Pro Gly Ser Trp Pro Trp Gln Val Ser

20 25 30

Leu Gln Asp Lys Thr Gly Phe His Phe Cys Gly Gly Ser Leu Ile Ser

35 40 45

Glu Asp Trp Val Val Thr Ala Ala His Cys Gly Val Arg Thr Ser Asp

50 55 60

Val Val Val Ala Gly Glu Phe Asp Gln Gly Ser Asp Glu Glu Asn Ile

65 70 75 80

Gln Val Leu Lys Ile Ala Lys Val Phe Lys Asn Pro Lys Phe Ser Ile

85 90 95

Leu Thr Val Asn Asn Asp Ile Thr Leu Leu Lys Leu Ala Thr Pro Ala

100 105 110

Arg Phe Ser Gln Thr Val Ser Ala Val Cys Leu Pro Ser Ala Asp Asp

115 120 125

Asp Phe Pro Ala Gly Thr Leu Cys Ala Thr Thr Gly Trp Gly Lys Thr

130 135 140

Lys Tyr Asn Ala Asn Lys Thr Pro Asp Lys Leu Gln Gln Ala Ala Leu

145 150 155 160

Pro Leu Leu Ser Asn Ala Glu Cys Lys Lys Ser Trp Gly Arg Arg Ile

165 170 175

Thr Asp Val Met Ile Cys Ala Gly Ala Ser Gly Val Ser Ser Cys Met

180 185 190

Gly Asp Ser Gly Gly Pro Leu Val Cys Gln Lys Asp Gly Ala Trp Thr

195 200 205

Leu Val Gly Ile Val Ser Trp Gly Ser Asp Thr Cys Ser Thr Ser Ser

210 215 220

Pro Gly Val Tyr Ala Arg Val Thr Lys Leu Ile Pro Trp Val Gln Lys

225 230 235 240

Ile Leu Ala Ala Asn

245

<210> 3

<211> 245

<212> PRT

<213> Artificial Sequence

<220>

<223> mutant 1

<400> 3

Cys Gly Val Pro Ala Ile His Pro Val Leu Ser Gly Leu Ser Arg Ile

1 5 10 15

Val Asn Gly Glu Asp Ala Val Pro Gly Ser Trp Pro Trp Gln Val Ser

20 25 30

Leu Gln Asp Lys Thr Gly Phe His Phe Cys Gly Gly Ser Leu Ile Ser

35 40 45

Glu Asp Trp Val Val Thr Ala Ala His Cys Gly Val Arg Thr Ser Asp

50 55 60

Val Val Val Ala Gly Glu Phe Asp Gln Gly Ser Asp Glu Glu Asn Ile

65 70 75 80

Gln Val Leu Lys Ile Ala Lys Val Phe Lys Asn Pro Lys Phe Ser Ile

85 90 95

Leu Thr Val Asn Asn Asp Ile Thr Leu Leu Lys Leu Ala Thr Pro Ala

100 105 110

Arg Ala Ser Gln Thr Val Ser Ala Val Cys Leu Pro Ser Ala Asp Asp

115 120 125

Asp Phe Pro Ala Gly Thr Leu Cys Ala Thr Thr Gly Trp Gly Lys Thr

130 135 140

Lys Tyr Asn Ala Asn Lys Thr Pro Asp Lys Leu Gln Gln Ala Ala Leu

145 150 155 160

Pro Leu Leu Ser Asn Ala Glu Cys Lys Lys Ser Trp Gly Arg Arg Ile

165 170 175

Thr Asp Val Met Ile Cys Ala Gly Ala Ser Gly Val Ser Ser Cys Met

180 185 190

Gly Asp Ser Gly Gly Pro Leu Val Cys Gln Lys Asp Gly Ala Trp Thr

195 200 205

Leu Val Gly Ile Val Ser Trp Gly Ser Asp Thr Cys Ser Thr Ser Ser

210 215 220

Pro Gly Val Tyr Ala Arg Val Thr Lys Leu Ile Pro Trp Val Gln Lys

225 230 235 240

Ile Leu Ala Ala Asn

245

<210> 4

<211> 245

<212> PRT

<213> Artificial Sequence

<220>

<223> mutant 2

<400> 4

Cys Gly Val Pro Ala Ile His Pro Val Leu Ser Gly Leu Ser Arg Ile

1 5 10 15

Val Asn Gly Glu Asp Ala Val Pro Gly Ser Trp Pro Trp Gln Val Ser

20 25 30

Leu Gln Asp Lys Thr Gly Phe His Phe Cys Gly Gly Ser Leu Ile Ser

35 40 45

Glu Asp Trp Val Val Thr Ala Ala His Cys Gly Val Arg Thr Ser Asp

50 55 60

Val Val Val Ala Gly Glu Phe Asp Gln Gly Ser Asp Glu Glu Asn Ile

65 70 75 80

Gln Val Leu Lys Ile Ala Lys Val Phe Lys Asn Pro Lys Phe Ser Ile

85 90 95

Leu Thr Val Asn Asn Asp Ile Thr Leu Leu Lys Leu Ala Thr Pro Ala

100 105 110

Arg Arg Ser Gln Thr Val Ser Ala Val Cys Leu Pro Ser Ala Asp Asp

115 120 125

Asp Phe Pro Ala Gly Thr Leu Cys Ala Thr Thr Gly Trp Gly Lys Thr

130 135 140

Lys Tyr Asn Ala Asn Lys Thr Pro Asp Lys Leu Gln Gln Ala Ala Leu

145 150 155 160

Pro Leu Leu Ser Asn Ala Glu Cys Lys Lys Ser Trp Gly Arg Arg Ile

165 170 175

Thr Asp Val Met Ile Cys Ala Gly Ala Ser Gly Val Ser Ser Cys Met

180 185 190

Gly Asp Ser Gly Gly Pro Leu Val Cys Gln Lys Asp Gly Ala Trp Thr

195 200 205

Leu Val Gly Ile Val Ser Trp Gly Ser Asp Thr Cys Ser Thr Ser Ser

210 215 220

Pro Gly Val Tyr Ala Arg Val Thr Lys Leu Ile Pro Trp Val Gln Lys

225 230 235 240

Ile Leu Ala Ala Asn

245

<210> 5

<211> 245

<212> PRT

<213> Artificial Sequence

<220>

<223> mutant 3

<400> 5

Cys Gly Val Pro Ala Ile His Pro Val Leu Ser Gly Leu Ser Arg Ile

1 5 10 15

Val Asn Gly Glu Asp Ala Val Pro Gly Ser Trp Pro Trp Gln Val Ser

20 25 30

Leu Gln Asp Lys Thr Gly Phe His Phe Cys Gly Gly Ser Leu Ile Ser

35 40 45

Glu Asp Trp Val Val Thr Ala Ala His Cys Gly Val Arg Thr Ser Asp

50 55 60

Val Val Val Ala Gly Glu Phe Asp Gln Gly Ser Asp Glu Glu Asn Ile

65 70 75 80

Gln Val Leu Lys Ile Ala Lys Val Phe Lys Asn Pro Lys Phe Ser Ile

85 90 95

Leu Thr Val Asn Asn Asp Ile Thr Leu Leu Lys Leu Ala Thr Pro Ala

100 105 110

Arg Asn Ser Gln Thr Val Ser Ala Val Cys Leu Pro Ser Ala Asp Asp

115 120 125

Asp Phe Pro Ala Gly Thr Leu Cys Ala Thr Thr Gly Trp Gly Lys Thr

130 135 140

Lys Tyr Asn Ala Asn Lys Thr Pro Asp Lys Leu Gln Gln Ala Ala Leu

145 150 155 160

Pro Leu Leu Ser Asn Ala Glu Cys Lys Lys Ser Trp Gly Arg Arg Ile

165 170 175

Thr Asp Val Met Ile Cys Ala Gly Ala Ser Gly Val Ser Ser Cys Met

180 185 190

Gly Asp Ser Gly Gly Pro Leu Val Cys Gln Lys Asp Gly Ala Trp Thr

195 200 205

Leu Val Gly Ile Val Ser Trp Gly Ser Asp Thr Cys Ser Thr Ser Ser

210 215 220

Pro Gly Val Tyr Ala Arg Val Thr Lys Leu Ile Pro Trp Val Gln Lys

225 230 235 240

Ile Leu Ala Ala Asn

245

<210> 6

<211> 245

<212> PRT

<213> Artificial Sequence

<220>

<223> mutant 4

<400> 6

Cys Gly Val Pro Ala Ile His Pro Val Leu Ser Gly Leu Ser Arg Ile

1 5 10 15

Val Asn Gly Glu Asp Ala Val Pro Gly Ser Trp Pro Trp Gln Val Ser

20 25 30

Leu Gln Asp Lys Thr Gly Phe His Phe Cys Gly Gly Ser Leu Ile Ser

35 40 45

Glu Asp Trp Val Val Thr Ala Ala His Cys Gly Val Arg Thr Ser Asp

50 55 60

Val Val Val Ala Gly Glu Phe Asp Gln Gly Ser Asp Glu Glu Asn Ile

65 70 75 80

Gln Val Leu Lys Ile Ala Lys Val Phe Lys Asn Pro Lys Phe Ser Ile

85 90 95

Leu Thr Val Asn Asn Asp Ile Thr Leu Leu Lys Leu Ala Thr Pro Ala

100 105 110

Arg Glu Ser Gln Thr Val Ser Ala Val Cys Leu Pro Ser Ala Asp Asp

115 120 125

Asp Phe Pro Ala Gly Thr Leu Cys Ala Thr Thr Gly Trp Gly Lys Thr

130 135 140

Lys Tyr Asn Ala Asn Lys Thr Pro Asp Lys Leu Gln Gln Ala Ala Leu

145 150 155 160

Pro Leu Leu Ser Asn Ala Glu Cys Lys Lys Ser Trp Gly Arg Arg Ile

165 170 175

Thr Asp Val Met Ile Cys Ala Gly Ala Ser Gly Val Ser Ser Cys Met

180 185 190

Gly Asp Ser Gly Gly Pro Leu Val Cys Gln Lys Asp Gly Ala Trp Thr

195 200 205

Leu Val Gly Ile Val Ser Trp Gly Ser Asp Thr Cys Ser Thr Ser Ser

210 215 220

Pro Gly Val Tyr Ala Arg Val Thr Lys Leu Ile Pro Trp Val Gln Lys

225 230 235 240

Ile Leu Ala Ala Asn

245

<210> 7

<211> 245

<212> PRT

<213> Artificial Sequence

<220>

<223> mutant 5

<400> 7

Cys Gly Val Pro Ala Ile His Pro Val Leu Ser Gly Leu Ser Arg Ile

1 5 10 15

Val Asn Gly Glu Asp Ala Val Pro Gly Ser Trp Pro Trp Gln Val Ser

20 25 30

Leu Gln Asp Lys Thr Gly Phe His Phe Cys Gly Gly Ser Leu Ile Ser

35 40 45

Glu Asp Trp Val Val Thr Ala Ala His Cys Gly Val Arg Thr Ser Asp

50 55 60

Val Val Val Ala Gly Glu Phe Asp Gln Gly Ser Asp Glu Glu Asn Ile

65 70 75 80

Gln Val Leu Lys Ile Ala Lys Val Phe Lys Asn Pro Lys Phe Ser Ile

85 90 95

Leu Thr Val Asn Asn Asp Ile Thr Leu Leu Lys Leu Ala Thr Pro Ala

100 105 110

Arg Gln Ser Gln Thr Val Ser Ala Val Cys Leu Pro Ser Ala Asp Asp

115 120 125

Asp Phe Pro Ala Gly Thr Leu Cys Ala Thr Thr Gly Trp Gly Lys Thr

130 135 140

Lys Tyr Asn Ala Asn Lys Thr Pro Asp Lys Leu Gln Gln Ala Ala Leu

145 150 155 160

Pro Leu Leu Ser Asn Ala Glu Cys Lys Lys Ser Trp Gly Arg Arg Ile

165 170 175

Thr Asp Val Met Ile Cys Ala Gly Ala Ser Gly Val Ser Ser Cys Met

180 185 190

Gly Asp Ser Gly Gly Pro Leu Val Cys Gln Lys Asp Gly Ala Trp Thr

195 200 205

Leu Val Gly Ile Val Ser Trp Gly Ser Asp Thr Cys Ser Thr Ser Ser

210 215 220

Pro Gly Val Tyr Ala Arg Val Thr Lys Leu Ile Pro Trp Val Gln Lys

225 230 235 240

Ile Leu Ala Ala Asn

245

<210> 8

<211> 245

<212> PRT

<213> Artificial Sequence

<220>

<223> mutant 6

<400> 8

Cys Gly Val Pro Ala Ile His Pro Val Leu Ser Gly Leu Ser Arg Ile

1 5 10 15

Val Asn Gly Glu Asp Ala Val Pro Gly Ser Trp Pro Trp Gln Val Ser

20 25 30

Leu Gln Asp Lys Thr Gly Phe His Phe Cys Gly Gly Ser Leu Ile Ser

35 40 45

Glu Asp Trp Val Val Thr Ala Ala His Cys Gly Val Arg Thr Ser Asp

50 55 60

Val Val Val Ala Gly Glu Phe Asp Gln Gly Ser Asp Glu Glu Asn Ile

65 70 75 80

Gln Val Leu Lys Ile Ala Lys Val Phe Lys Asn Pro Lys Phe Ser Ile

85 90 95

Leu Thr Val Asn Asn Asp Ile Thr Leu Leu Lys Leu Ala Thr Pro Ala

100 105 110

Arg His Ser Gln Thr Val Ser Ala Val Cys Leu Pro Ser Ala Asp Asp

115 120 125

Asp Phe Pro Ala Gly Thr Leu Cys Ala Thr Thr Gly Trp Gly Lys Thr

130 135 140

Lys Tyr Asn Ala Asn Lys Thr Pro Asp Lys Leu Gln Gln Ala Ala Leu

145 150 155 160

Pro Leu Leu Ser Asn Ala Glu Cys Lys Lys Ser Trp Gly Arg Arg Ile

165 170 175

Thr Asp Val Met Ile Cys Ala Gly Ala Ser Gly Val Ser Ser Cys Met

180 185 190

Gly Asp Ser Gly Gly Pro Leu Val Cys Gln Lys Asp Gly Ala Trp Thr

195 200 205

Leu Val Gly Ile Val Ser Trp Gly Ser Asp Thr Cys Ser Thr Ser Ser

210 215 220

Pro Gly Val Tyr Ala Arg Val Thr Lys Leu Ile Pro Trp Val Gln Lys

225 230 235 240

Ile Leu Ala Ala Asn

245

<210> 9

<211> 245

<212> PRT

<213> Artificial Sequence

<220>

<223> mutant 7

<400> 9

Cys Gly Val Pro Ala Ile His Pro Val Leu Ser Gly Leu Ser Arg Ile

1 5 10 15

Val Asn Gly Glu Asp Ala Val Pro Gly Ser Trp Pro Trp Gln Val Ser

20 25 30

Leu Gln Asp Lys Thr Gly Phe His Phe Cys Gly Gly Ser Leu Ile Ser

35 40 45

Glu Asp Trp Val Val Thr Ala Ala His Cys Gly Val Arg Thr Ser Asp

50 55 60

Val Val Val Ala Gly Glu Phe Asp Gln Gly Ser Asp Glu Glu Asn Ile

65 70 75 80

Gln Val Leu Lys Ile Ala Lys Val Phe Lys Asn Pro Lys Phe Ser Ile

85 90 95

Leu Thr Val Asn Asn Asp Ile Thr Leu Leu Lys Leu Ala Thr Pro Ala

100 105 110

Arg Ile Ser Gln Thr Val Ser Ala Val Cys Leu Pro Ser Ala Asp Asp

115 120 125

Asp Phe Pro Ala Gly Thr Leu Cys Ala Thr Thr Gly Trp Gly Lys Thr

130 135 140

Lys Tyr Asn Ala Asn Lys Thr Pro Asp Lys Leu Gln Gln Ala Ala Leu

145 150 155 160

Pro Leu Leu Ser Asn Ala Glu Cys Lys Lys Ser Trp Gly Arg Arg Ile

165 170 175

Thr Asp Val Met Ile Cys Ala Gly Ala Ser Gly Val Ser Ser Cys Met

180 185 190

Gly Asp Ser Gly Gly Pro Leu Val Cys Gln Lys Asp Gly Ala Trp Thr

195 200 205

Leu Val Gly Ile Val Ser Trp Gly Ser Asp Thr Cys Ser Thr Ser Ser

210 215 220

Pro Gly Val Tyr Ala Arg Val Thr Lys Leu Ile Pro Trp Val Gln Lys

225 230 235 240

Ile Leu Ala Ala Asn

245

<210> 10

<211> 245

<212> PRT

<213> Artificial Sequence

<220>

<223> mutant 8

<400> 10

Cys Gly Val Pro Ala Ile His Pro Val Leu Ser Gly Leu Ser Arg Ile

1 5 10 15

Val Asn Gly Glu Asp Ala Val Pro Gly Ser Trp Pro Trp Gln Val Ser

20 25 30

Leu Gln Asp Lys Thr Gly Phe His Phe Cys Gly Gly Ser Leu Ile Ser

35 40 45

Glu Asp Trp Val Val Thr Ala Ala His Cys Gly Val Arg Thr Ser Asp

50 55 60

Val Val Val Ala Gly Glu Phe Asp Gln Gly Ser Asp Glu Glu Asn Ile

65 70 75 80

Gln Val Leu Lys Ile Ala Lys Val Phe Lys Asn Pro Lys Phe Ser Ile

85 90 95

Leu Thr Val Asn Asn Asp Ile Thr Leu Leu Lys Leu Ala Thr Pro Ala

100 105 110

Arg Lys Ser Gln Thr Val Ser Ala Val Cys Leu Pro Ser Ala Asp Asp

115 120 125

Asp Phe Pro Ala Gly Thr Leu Cys Ala Thr Thr Gly Trp Gly Lys Thr

130 135 140

Lys Tyr Asn Ala Asn Lys Thr Pro Asp Lys Leu Gln Gln Ala Ala Leu

145 150 155 160

Pro Leu Leu Ser Asn Ala Glu Cys Lys Lys Ser Trp Gly Arg Arg Ile

165 170 175

Thr Asp Val Met Ile Cys Ala Gly Ala Ser Gly Val Ser Ser Cys Met

180 185 190

Gly Asp Ser Gly Gly Pro Leu Val Cys Gln Lys Asp Gly Ala Trp Thr

195 200 205

Leu Val Gly Ile Val Ser Trp Gly Ser Asp Thr Cys Ser Thr Ser Ser

210 215 220

Pro Gly Val Tyr Ala Arg Val Thr Lys Leu Ile Pro Trp Val Gln Lys

225 230 235 240

Ile Leu Ala Ala Asn

245

<210> 11

<211> 245

<212> PRT

<213> Artificial Sequence

<220>

<223> mutant 9

<400> 11

Cys Gly Val Pro Ala Ile His Pro Val Leu Ser Gly Leu Ser Arg Ile

1 5 10 15

Val Asn Gly Glu Asp Ala Val Pro Gly Ser Trp Pro Trp Gln Val Ser

20 25 30

Leu Gln Asp Lys Thr Gly Phe His Phe Cys Gly Gly Ser Leu Ile Ser

35 40 45

Glu Asp Trp Val Val Thr Ala Ala His Cys Gly Val Arg Thr Ser Asp

50 55 60

Val Val Val Ala Gly Glu Phe Asp Gln Gly Ser Asp Glu Glu Asn Ile

65 70 75 80

Gln Val Leu Lys Ile Ala Lys Val Phe Lys Asn Pro Lys Phe Ser Ile

85 90 95

Leu Thr Val Asn Asn Asp Ile Thr Leu Leu Lys Leu Ala Thr Pro Ala

100 105 110

Arg Pro Ser Gln Thr Val Ser Ala Val Cys Leu Pro Ser Ala Asp Asp

115 120 125

Asp Phe Pro Ala Gly Thr Leu Cys Ala Thr Thr Gly Trp Gly Lys Thr

130 135 140

Lys Tyr Asn Ala Asn Lys Thr Pro Asp Lys Leu Gln Gln Ala Ala Leu

145 150 155 160

Pro Leu Leu Ser Asn Ala Glu Cys Lys Lys Ser Trp Gly Arg Arg Ile

165 170 175

Thr Asp Val Met Ile Cys Ala Gly Ala Ser Gly Val Ser Ser Cys Met

180 185 190

Gly Asp Ser Gly Gly Pro Leu Val Cys Gln Lys Asp Gly Ala Trp Thr

195 200 205

Leu Val Gly Ile Val Ser Trp Gly Ser Asp Thr Cys Ser Thr Ser Ser

210 215 220

Pro Gly Val Tyr Ala Arg Val Thr Lys Leu Ile Pro Trp Val Gln Lys

225 230 235 240

Ile Leu Ala Ala Asn

245

<210> 12

<211> 245

<212> PRT

<213> Artificial Sequence

<220>

<223> mutant 10

<400> 12

Cys Gly Val Pro Ala Ile His Pro Val Leu Ser Gly Leu Ser Arg Ile

1 5 10 15

Val Asn Gly Glu Asp Ala Val Pro Gly Ser Trp Pro Trp Gln Val Ser

20 25 30

Leu Gln Asp Lys Thr Gly Phe His Phe Cys Gly Gly Ser Leu Ile Ser

35 40 45

Glu Asp Trp Val Val Thr Ala Ala His Cys Gly Val Arg Thr Ser Asp

50 55 60

Val Val Val Ala Gly Glu Phe Asp Gln Gly Ser Asp Glu Glu Asn Ile

65 70 75 80

Gln Val Leu Lys Ile Ala Lys Val Phe Lys Asn Pro Lys Phe Ser Ile

85 90 95

Leu Thr Val Asn Asn Asp Ile Thr Leu Leu Lys Leu Ala Thr Pro Ala

100 105 110

Arg Ser Ser Gln Thr Val Ser Ala Val Cys Leu Pro Ser Ala Asp Asp

115 120 125

Asp Phe Pro Ala Gly Thr Leu Cys Ala Thr Thr Gly Trp Gly Lys Thr

130 135 140

Lys Tyr Asn Ala Asn Lys Thr Pro Asp Lys Leu Gln Gln Ala Ala Leu

145 150 155 160

Pro Leu Leu Ser Asn Ala Glu Cys Lys Lys Ser Trp Gly Arg Arg Ile

165 170 175

Thr Asp Val Met Ile Cys Ala Gly Ala Ser Gly Val Ser Ser Cys Met

180 185 190

Gly Asp Ser Gly Gly Pro Leu Val Cys Gln Lys Asp Gly Ala Trp Thr

195 200 205

Leu Val Gly Ile Val Ser Trp Gly Ser Asp Thr Cys Ser Thr Ser Ser

210 215 220

Pro Gly Val Tyr Ala Arg Val Thr Lys Leu Ile Pro Trp Val Gln Lys

225 230 235 240

Ile Leu Ala Ala Asn

245

<210> 13

<211> 245

<212> PRT

<213> Artificial Sequence

<220>

<223> mutant 11

<400> 13

Cys Gly Val Pro Ala Ile His Pro Val Leu Ser Gly Leu Ser Arg Ile

1 5 10 15

Val Asn Gly Glu Asp Ala Val Pro Gly Ser Trp Pro Trp Gln Val Ser

20 25 30

Leu Gln Asp Lys Thr Gly Phe His Phe Cys Gly Gly Ser Leu Ile Ser

35 40 45

Glu Asp Trp Val Val Thr Ala Ala His Cys Gly Val Arg Thr Ser Asp

50 55 60

Val Val Val Ala Gly Glu Phe Asp Gln Gly Ser Asp Glu Glu Asn Ile

65 70 75 80

Gln Val Leu Lys Ile Ala Lys Val Phe Lys Asn Pro Lys Phe Ser Ile

85 90 95

Leu Thr Val Asn Asn Asp Ile Thr Leu Leu Lys Leu Ala Thr Pro Ala

100 105 110

Arg Thr Ser Gln Thr Val Ser Ala Val Cys Leu Pro Ser Ala Asp Asp

115 120 125

Asp Phe Pro Ala Gly Thr Leu Cys Ala Thr Thr Gly Trp Gly Lys Thr

130 135 140

Lys Tyr Asn Ala Asn Lys Thr Pro Asp Lys Leu Gln Gln Ala Ala Leu

145 150 155 160

Pro Leu Leu Ser Asn Ala Glu Cys Lys Lys Ser Trp Gly Arg Arg Ile

165 170 175

Thr Asp Val Met Ile Cys Ala Gly Ala Ser Gly Val Ser Ser Cys Met

180 185 190

Gly Asp Ser Gly Gly Pro Leu Val Cys Gln Lys Asp Gly Ala Trp Thr

195 200 205

Leu Val Gly Ile Val Ser Trp Gly Ser Asp Thr Cys Ser Thr Ser Ser

210 215 220

Pro Gly Val Tyr Ala Arg Val Thr Lys Leu Ile Pro Trp Val Gln Lys

225 230 235 240

Ile Leu Ala Ala Asn

245

<210> 14

<211> 245

<212> PRT

<213> Artificial Sequence

<220>

<223> mutant 12

<400> 14

Cys Gly Val Pro Ala Ile His Pro Val Leu Ser Gly Leu Ser Arg Ile

1 5 10 15

Val Asn Gly Glu Asp Ala Val Pro Gly Ser Trp Pro Trp Gln Val Ser

20 25 30

Leu Gln Asp Lys Thr Gly Phe His Phe Cys Gly Gly Ser Leu Ile Ser

35 40 45

Glu Asp Trp Val Val Thr Ala Ala His Cys Gly Val Arg Thr Ser Asp

50 55 60

Val Val Val Ala Gly Glu Phe Asp Gln Gly Ser Asp Glu Glu Asn Ile

65 70 75 80

Gln Val Leu Lys Ile Ala Lys Val Phe Lys Asn Pro Lys Phe Ser Ile

85 90 95

Leu Thr Val Asn Asn Asp Ile Thr Leu Leu Lys Leu Ala Thr Pro Ala

100 105 110

Arg Val Ser Gln Thr Val Ser Ala Val Cys Leu Pro Ser Ala Asp Asp

115 120 125

Asp Phe Pro Ala Gly Thr Leu Cys Ala Thr Thr Gly Trp Gly Lys Thr

130 135 140

Lys Tyr Asn Ala Asn Lys Thr Pro Asp Lys Leu Gln Gln Ala Ala Leu

145 150 155 160

Pro Leu Leu Ser Asn Ala Glu Cys Lys Lys Ser Trp Gly Arg Arg Ile

165 170 175

Thr Asp Val Met Ile Cys Ala Gly Ala Ser Gly Val Ser Ser Cys Met

180 185 190

Gly Asp Ser Gly Gly Pro Leu Val Cys Gln Lys Asp Gly Ala Trp Thr

195 200 205

Leu Val Gly Ile Val Ser Trp Gly Ser Asp Thr Cys Ser Thr Ser Ser

210 215 220

Pro Gly Val Tyr Ala Arg Val Thr Lys Leu Ile Pro Trp Val Gln Lys

225 230 235 240

Ile Leu Ala Ala Asn

245

<210> 15

<211> 13

<212> PRT

<213> Artificial Sequence

<220>

<223> A chain

<400> 15

Cys Gly Val Pro Ala Ile His Pro Val Leu Ser Gly Leu

1 5 10

<210> 16

<211> 131

<212> PRT

<213> Artificial Sequence

<220>

<223> B chain

<400> 16

Ile Val Asn Gly Glu Asp Ala Val Pro Gly Ser Trp Pro Trp Gln Val

1 5 10 15

Ser Leu Gln Asp Lys Thr Gly Phe His Phe Cys Gly Gly Ser Leu Ile

20 25 30

Ser Glu Asp Trp Val Val Thr Ala Ala His Cys Gly Val Arg Thr Ser

35 40 45

Asp Val Val Val Ala Gly Glu Phe Asp Gln Gly Ser Asp Glu Glu Asn

50 55 60

Ile Gln Val Leu Lys Ile Ala Lys Val Phe Lys Asn Pro Lys Phe Ser

65 70 75 80

Ile Leu Thr Val Asn Asn Asp Ile Thr Leu Leu Lys Leu Ala Thr Pro

85 90 95

Ala Arg Phe Ser Gln Thr Val Ser Ala Val Cys Leu Pro Ser Ala Asp

100 105 110

Asp Asp Phe Pro Ala Gly Thr Leu Cys Ala Thr Thr Gly Trp Gly Lys

115 120 125

Thr Lys Tyr

130

<210> 17

<211> 97

<212> PRT

<213> Artificial Sequence

<220>

<223> C chain

<400> 17

Asn Lys Thr Pro Asp Lys Leu Gln Gln Ala Ala Leu Pro Leu Leu Ser

1 5 10 15

Asn Ala Glu Cys Lys Lys Ser Trp Gly Arg Arg Ile Thr Asp Val Met

20 25 30

Ile Cys Ala Gly Ala Ser Gly Val Ser Ser Cys Met Gly Asp Ser Gly

35 40 45

Gly Pro Leu Val Cys Gln Lys Asp Gly Ala Trp Thr Leu Val Gly Ile

50 55 60

Val Ser Trp Gly Ser Asp Thr Cys Ser Thr Ser Ser Pro Gly Val Tyr

65 70 75 80

Ala Arg Val Thr Lys Leu Ile Pro Trp Val Gln Lys Ile Leu Ala Ala

85 90 95

Asn

Claims (12)

1. The mutant of the human chymotrypsinogen is characterized in that the amino acid sequence of the human chymotrypsinogen is shown as SEQ ID NO. 2; the mutant is obtained after the 114 th position of the human chymotrypsinogen is replaced, and the 114 th position is replaced by alanine, arginine, asparagine, glutamic acid, glutamine, histidine, lysine, proline, serine, threonine or valine;

the mutant comprises at least the function of the human chymotrypsinogen.

2. The mutant human chymotrypsinogen of claim 1, wherein the substitution at position 114 is proline or threonine.

3. A human chymotrypsin prepared from a mutant of human chymotrypsinogen as claimed in claim 1 or 2.

4. The human chymotrypsin according to claim 3, wherein the amino acid sequences of the 3 strands of the human chymotrypsin are shown in SEQ ID NO 15-17, respectively.

5. A nucleotide encoding the mutant of human chymotrypsinogen according to claim 1 or 2 or the human chymotrypsin according to claim 3 or 4.

6. An expression vector comprising the nucleotide of claim 5;

preferably, in the expression vector, when the nucleotide is a nucleotide encoding the human chymotrypsinogen mutant, the 5' end of the nucleotide is a nucleotide encoding a signal peptide;

the amino acid of the signal peptide is preferably shown as SEQ ID NO. 1 in the sequence table.

7. A host cell comprising the expression vector of claim 6; the host cell is preferably an E.coli cell or a yeast cell.

8. A method of preparing human chymotrypsin, comprising: activating a mutant of human chymotrypsinogen as defined in claim 1 or 2;

preferably, the method further comprises: culturing the host cell of claim 7 to obtain said mutant of human chymotrypsinogen prior to said activation.

9. Use of a mutant of human chymotrypsinogen according to claim 1 or 2 or human chymotrypsin according to claim 3 or 4 for the degradation of proteins.

10. Use of the mutant of human chymotrypsinogen according to claim 1 or 2 or the human chymotrypsin according to claim 3 or 4 in the preparation of a medicament for the treatment of a disease;

preferably, the disease is selected from the group consisting of surgical inflammation, trauma, hematoma and abscess, post-operative inflammation, organ inflammatory diseases and disorders.

11. A composition comprising the human chymotrypsin of claim 3 or 4; preferably, the composition further comprises a pharmaceutically acceptable carrier or excipient.

12. A kit of parts comprising kit a and kit B, wherein kit a comprises a mutant of human chymotrypsinogen according to claim 1 or 2 or human chymotrypsin according to claim 3 or 4; preferably, the medicine box B comprises expectorant, antibiotics, hormone, and other Chinese medicines, enzymes and growth factors with debridement, antiphlogosis and bacteriostasis effects;

when the kit a comprises the mutant of human chymotrypsinogen of claim 1 or 2, the kit of parts further comprises kit C comprising trypsin for activating the mutant of human chymotrypsinogen.

Images