CN109439643B - Novel lysine specific endonuclease and preparation method thereof - Google Patents
Novel lysine specific endonuclease and preparation method thereof Download PDFInfo
- Publication number
- CN109439643B CN109439643B CN201811394022.5A CN201811394022A CN109439643B CN 109439643 B CN109439643 B CN 109439643B CN 201811394022 A CN201811394022 A CN 201811394022A CN 109439643 B CN109439643 B CN 109439643B
- Authority
- CN
- China
- Prior art keywords
- gly
- ser
- ala
- lysine
- specific endonuclease
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
- C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
- C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
- C12N9/6402—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from non-mammals
- C12N9/6405—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from non-mammals not being snakes
- C12N9/6408—Serine endopeptidases (3.4.21)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/70—Vectors or expression systems specially adapted for E. coli
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
- C12Y304/21—Serine endopeptidases (3.4.21)
- C12Y304/2105—Lysyl endopeptidase (3.4.21.50)
Landscapes
- Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Organic Chemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Biomedical Technology (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Biotechnology (AREA)
- Biochemistry (AREA)
- Microbiology (AREA)
- Molecular Biology (AREA)
- Medicinal Chemistry (AREA)
- Physics & Mathematics (AREA)
- Biophysics (AREA)
- Plant Pathology (AREA)
- Enzymes And Modification Thereof (AREA)
Abstract
The invention discloses a novel lysine specific endonuclease, which can detect nanogram-level residual enzyme in a sample by an ELISA method and ensure the activity of the holoenzyme of the wild lysine specific endonuclease through a proper flexible connecting peptide ligase core peptide and a histidine tag. Meanwhile, the invention also provides a preparation method of the enzyme, which has the beneficial effect of high yield through genetic engineering modification and fermentation process optimization, and the recovery amount of 857mg of novel lysine specific endonuclease pure product can be reached after 1L of fermentation liquor is purified, so that the enzyme has the potential of industrialization.
Description
Technical Field
The invention relates to the field of genetic engineering, in particular to a novel lysine specific endonuclease and a preparation method thereof.
Background
Lysine-specific endonuclease (EC 3.4.21.50), also called Lysyl endopeptidase, achromobacter I, lysine endopeptidase, Lysyl endopeptidase, lysine C-terminal endopeptidase, Lys-C endopeptidase, is a serine protease, and was originally found and isolated from soil bacteria by Masaki et al. The lysine-specific endonuclease has high specificity and can specifically cleave peptide bonds at the carboxyl terminals of lysine residues and S-aminoethylcysteine residues in peptide chains.
The lysine specific endonuclease also has the following remarkable characteristics: 1) is 10 times higher than the activity of bovine trypsin; 2) has wide pH tolerance (pH 8.5-10.5); 3) stronger surfactant tolerance, such as maintaining holoenzyme activity in solutions containing 4mol/L urea or 0.1% SDS; 4) the trypsin has higher substrate specificity than the trypsin, the trypsin can recognize and cut lysine and arginine, and the lysine-specific endonuclease usually only recognizes and cuts lysine, so the enzyme cutting conversion rate and the product purity can be obviously improved. Due to the characteristics, the lysine specific endonuclease has important application value in the fields of protein sequence analysis (such as mass spectrometry), proteomics research (such as peptide mapping analysis), biopharmaceuticals (such as enzyme digestion of proinsulin), Lys-X compound enzyme catalytic synthesis and the like.
The currently used lysine-specific endonucleases have the following problems and disadvantages:
1. when the wild strain is used for expressing the lysine specific endonuclease, the expression level is too low, and the industrial requirement cannot be met. Although the expression level is improved to some extent by using the mutant strain obtained by wild mutagenesis breeding, the stability of the mutant strain is not high, and the risk of back mutation exists. CN103865836A "a mutant strain of lysobacter enzymogenes and a preparation method thereof" discloses a mutant strain L.enzymogenes TGJZC-041 which is obtained by natural breeding to obtain a lysobacter enzymogenes strain L.enzymogenes PGJZC30 and then space mutation breeding and has the highest expression level. The strain is fermented and cultured for 3 days by using a 50L fermentation tank, the expression amount is 1.3U/mL, and 390mg of Lys-C is obtained after purification, namely the yield is 7.8 mg/L.
2. In order to overcome the defect of low expression level of wild bacteria, people use engineering strains such as escherichia coli and the like to prepare the recombinant lysine specific endonuclease, but the yield is not obviously improved, and the specific activity of the recombinant lysine specific endonuclease is reduced. US5248599A and EP0387646B1 disclose a method for preparing lysine-specific endonuclease using recombinant E.coli. The method uses escherichia coli to express lysine specific endonuclease zymogen, and uses an escherichia coli self-modifying and processing system to self-activate the zymogen to form mature enzyme with activity, and 1.6mg of recombinant lysine specific endonuclease pure product can be obtained from 1L of fermentation liquor. CN105950593A 'prokaryotic recombinant expression and preparation method of lysine specific endonuclease' discloses a method for preparing recombinant lysine specific endonuclease by using Escherichia coli. The method uses recombinant Escherichia coli to express recombinant lysine specific endonuclease zymogen, the product exists in the form of inclusion body, and the pure product of the recombinant lysine specific endonuclease is obtained through denaturation, renaturation, activation, ammonium sulfate precipitation, affinity chromatography, ultrafiltration and gel filtration chromatography. 1888mg zymogen can be obtained by denaturation of inclusion bodies obtained from each 1L fermentation system, 36.61mg of pure products of the recombinant lysine specificity endonuclease can be obtained by purification, and the recovery rate is 1.9%.
3. When the traditional lysyl-specific endonuclease is used in the field of biological pharmacy, the residual quantity of the lysine-specific endonuclease in the medicine cannot be detected by a high-sensitivity method. Detecting the residual quantity of lysine-specific endonuclease in the intermediate and the bulk drug by adopting a high performance liquid chromatography, wherein the detection limit can only reach the mu g level, and a degradation band of the lysine-specific endonuclease can not be detected; the protein electrophoresis method can detect the degradation band of the lysine specific endonuclease, but the detection limit can only reach the mu g level; the development cost of the lysine specificity endonuclease ELISA detection kit is high, and at present, no commercialized ELISA kit can be used for detecting the residue of lysine endopeptidase.
Therefore, it is very necessary to develop a novel lysine-specific endonuclease.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provide a lysine specific endonuclease and a preparation method thereof.
The technical scheme adopted by the invention is as follows:
a lysine-specific endonuclease, the structure of which is: lysine-specific endonuclease core-linker peptide-histidine tag.
As a further improvement of the above lysine-specific endonuclease, the lysine-specific endonuclease is one selected from the group consisting of a lysine-specific endonuclease derived from Pseudomonas aeruginosa, a lysine-specific endonuclease derived from Achromobacter leuco or a lysine-specific endonuclease derived from Lysobacter enzymogenes;
as a further improvement of the lysine specific endonuclease, the connecting peptide is a flexible connecting peptide and is selected from one of GGGGS, GGGGSGGGGS, GGGGSGGGGSGGS, GGSGGGSGGGGS and GGSGGGGSGGGGGS;
as a further improvement of the above lysine-specific endonuclease, the histidine tag is 4 to 8 consecutive histidines, preferably 6 histidines;
as a further improvement of the lysine-specific endonuclease, the amino acid sequence of the lysine-specific endonuclease is shown in SEQ ID NO: 4, respectively.
A preparation method of the lysine-acyl endonuclease comprises the following steps:
1) constructing a recombinant vector containing the lysine specificity endonuclease coding gene;
2) introducing the recombinant vector into an escherichia coli host cell to obtain a recombinant escherichia coli cell;
3) carrying out fermentation culture on the recombinant escherichia coli cells, adding an inducer, and collecting the recombinant escherichia coli cells after induction expression;
4) carrying out high-pressure homogenization and crushing on the recombinant escherichia coli cells obtained in the step 3) to obtain a cell suspension containing the novel lysine specific endonuclease;
5) purifying and freeze-drying the cell suspension containing the novel lysine specific endonuclease to obtain lysine specific endonuclease powder.
Wherein the recombinant vector in step 1) comprises a T7 promoter or a T7lac promoter, a polynucleotide molecule encoding the lysine-specific endonuclease of claim 1, and a T7 terminator located downstream of the polynucleotide molecule, wherein the polynucleotide molecule is located downstream of the promoter;
wherein the inducer in step 3) is 0.3mM IPTG;
wherein, the purification method in the step 5) is high-pressure homogenization and nickel column affinity chromatography.
The invention has the beneficial effects that:
the invention provides a novel lysine specific endonuclease, wherein the C-terminal of the endonuclease is provided with a histidine tag, and the enzyme with nanogram-level residues in a sample can be detected by a His tag ELISA detection kit, so that the novel lysine specific endonuclease is particularly suitable for the field of biological pharmacy; the enzyme core peptide and the histidine tag are connected through a proper flexible connecting peptide, so that the function of the histidine tag is retained, and the novel lysine specific endonuclease is ensured to have the whole enzyme activity of the wild lysine specific endonuclease.
The invention also provides a preparation method of the novel lysine specific endonuclease, the method has the beneficial effect of high yield, when the novel lysine specific endonuclease is cultured in a 7.5L fermentation tank, the expression level can reach 18000AU/L, and 857mg of novel lysine specific endonuclease pure product is obtained after 1L fermentation liquor is purified and is higher than the level reported in the current literature.
Drawings
FIG. 1 is a pGBCETS plasmid map;
FIG. 2 is an electrophoresis diagram of a fermentation sample of the lysine-specific endonuclease, in which arrows indicate that the novel lysine-specific endonuclease is 29.2kDa, and the second lane is a protein molecular weight standard which is 200kDa, 116kDa, 97.2kDa, 66.4kDa, 44.3kDa, 29kDa, 20.1kDa, 14.3kDa and 6.5kDa in sequence from top to bottom;
FIG. 3 shows the results of detecting lysine-specific endonuclease with His-tag ELISA kit;
FIG. 4 is a comparison graph of the effect of using wild-type lysine-specific endonuclease and novel lysine-specific endonuclease to cleave the insulin aspart precursor, wherein A is the insulin aspart intermediate prepared by wild-type lysine-specific endonuclease, B is the insulin aspart intermediate prepared by novel lysine-specific endonuclease, Peak 1 is the insulin aspart precursor, Peak 2 is the insulin aspart intermediate.
Detailed Description
The invention obtains fermentation liquor containing novel lysine specific incision enzyme by constructing genetic engineering strains and carrying out high-density fermentation, and then carries out detection and analysis on the enzyme activity.
1) Constructing a genetic engineering strain:
artificially synthesizing a coding gene of a novel lysine-specific endonuclease, carrying out enzyme digestion by using restriction endonucleases NdeI and EcoRI (purchased from NEB company), recovering a gene fragment by using an agarose gel DNA recovery kit (purchased from TIANGEN), then connecting the gene fragment with an NdeI and EcoRI digested expression vector pGBCETS (figure 1), transforming Escherichia coli TOP10 to obtain a transformant, and carrying out sequencing identification by using universal primers T7 promoter primer and T7 terminator primer to obtain a recombinant expression vector. The recombinant expression vector is transformed into escherichia coli BL21(DE3) by a calcium chloride method to obtain a novel lysine-specific endonuclease gene engineering strain.
2) High-density fermentation:
the genetic engineering strain is inoculated into a 7.5L fermentation tank containing 3L M9 culture medium (4g/L glucose, 1mM magnesium sulfate, 0.5g/L sodium chloride, 1g/L ammonium chloride, 3g/L potassium dihydrogen phosphate and 6g/L disodium hydrogen phosphate heptahydrate), the culture is carried out at 37 ℃ and the pH value is 7.0, after the glycerol is exhausted, a glycerol solution with the mass volume fraction of 50% is supplemented, when the dissolved oxygen is less than 15%, the stirring rate and the ventilation volume are increased, after 20h of culture, 214.47mg ITPG is added for induction expression, and after 20h of induction, the culture is carried out in the tank.
3) Enzyme activity detection and analysis:
the method specifically cuts a substrate according to lysyl specific endonuclease under a certain condition, so that p-nitroaniline in the substrate is cut, free p-nitroaniline develops color under visible light, the color of the free p-nitroaniline is in direct proportion to the concentration of the p-nitroaniline within a certain concentration range, and the enzyme activity of the lysyl specific endonuclease in a test sample is determined by a colorimetric method. Definition of enzyme activity: the amount of enzyme catalyzing the substrate to form 1. mu. mol of p-nitroaniline per minute at 30 ℃ and pH9.5 was 1 AU. The detection is carried out according to the following method:
blank is marked as A1 and fermentation broke is marked as A2. To A1 and A2 were added 2.6ml of 0.2mol/L AMP (2-Amino-2-menthyl-1,3-propanediol) buffer solution and 0.3ml of 2.5mmol/L substrate solution (Bz-lys-pNA), respectively, and the mixture was preheated in a 30 ℃ water bath for 5 minutes. 0.1ml of 180mmol/L Tris-HCl buffer solution was added to A1, 0.1ml of fermentation disruption solution was added to A2, and after rapid mixing, the mixture was reacted in a 30 ℃ water bath for 25 min. 1ml of 45% acetic acid was immediately added to A1 and A2 to terminate the reaction. A1 was zeroed as a blank and absorbance was measured at 405 nm.
The calculation formula of the enzyme activity of the method is as follows:
wherein, a represents the light absorption value of the sample, b represents the blank light absorption value, 9.62 represents the millimole extinction coefficient of the paranitroaniline, and c represents the dilution factor of the sample.
Example 1
Artificially synthesizing a coding gene of a novel lysyl specific endonuclease (SEQ ID NO: 1), wherein a gene engineering strain is constructed according to the method without adding flexible connecting peptide, and the gene engineering strain is cultured at high density and detected and analyzed by enzyme activity. The enzyme activity of the lysine specific endonuclease in the sample is 8600 AU/L.
Example 2
Artificially synthesizing a coding gene of a novel lysyl specific endonuclease (SEQ ID NO: 2), wherein the flexible connecting peptide is selected as GGGGS, constructing a genetic engineering strain according to the method, performing high-density culture, and detecting and analyzing enzyme activity. The enzyme activity of the lysine specific endonuclease in the sample is 10300 AU/L.
Example 3
Artificially synthesizing a coding gene of a novel lysyl specific endonuclease (SEQ ID NO: 3), wherein the flexible connecting peptide is selected as GGGGSGGGGS, constructing a genetic engineering strain according to the method, carrying out high-density culture, and carrying out enzyme activity detection and analysis. The enzyme activity of the lysine specific endonuclease in the sample is 18600 AU/L.
Example 4
Artificially synthesizing a coding gene of a novel lysyl specific endonuclease (SEQ ID NO: 4), wherein the flexible connecting peptide is selected as GGGGSGGGGSGGS, constructing a genetic engineering strain according to the method, carrying out high-density culture, and carrying out enzyme activity detection and analysis. The enzyme activity of the lysine specific endonuclease in the sample is 19200AU/L through detection.
Example 5
Artificially synthesizing a coding gene of a novel lysyl specific endonuclease (SEQ ID NO: 5), wherein the flexible connecting peptide is selected as GGSGGGSGGGGS, constructing a genetic engineering strain according to the method, carrying out high-density culture, and carrying out enzyme activity detection and analysis. The enzyme activity of the lysine specific endonuclease in the sample obtained by detection is 17400 AU/L.
Example 6
Artificially synthesizing a coding gene of a novel lysyl specific endonuclease (SEQ ID NO: 6), wherein the flexible connecting peptide is selected as GGSGGGSGGGGSGGGGS, constructing a genetic engineering strain according to the method, carrying out high-density culture, and carrying out enzyme activity detection and analysis. The enzyme activity of the lysine specific endonuclease in the sample obtained by detection is 17600 AU/L.
Example 7
Preparation of a novel lysine-specific endonuclease:
inoculating the new lysine specific endonuclease (SEQ ID NO: 3) gene engineering strain into a 20L fermentation tank containing 9L fermentation medium, performing fermentation culture according to the high-density fermentation method, and discharging the strain into the tank when the expression level reaches the maximum. Homogenizing the fermentation liquor once under the pressure of 90MPa by using a high-pressure homogenizer, loading the fermentation liquor to a UniIDA-80Ni chromatographic column, eluting the fermentation liquor by using a buffer solution (20mM PBS, 0.5M NaCl, 500mM imidazole, pH7.6 and the balance of water), collecting and obtaining a pure product of the novel lysine specific endonuclease, and carrying out vacuum freeze-drying to obtain the novel lysine specific endonuclease freeze-dried powder. The specific activity of the novel lysine-specific endonuclease was measured to be 2.53AU/mg as described in example 1.
Example 8
Preparation of wild-type lysine-specific endonuclease:
the wild-type lysine-specific endonuclease genetically engineered strain (SEQ ID NO: 7) was inoculated into a 20L fermentor containing 9L of fermentation medium, and fermentation culture was carried out as described in example 1, and the strain was placed in the fermentor when the expression level reached the maximum. Homogenizing the fermentation liquor once under the pressure of 90MPa by using a high-pressure homogenizer, obtaining a pure wild lysine specific endonuclease product by hydrophobic chromatography, and obtaining the wild lysine specific endonuclease freeze-dried powder after vacuum freeze-drying. The specific activity of the wild-type lysine-specific endonuclease was measured to be 2.44AU/mg as described in example 1.
Example 9
Detection of lysine-specific endonuclease with His-tag ELISA kit:
50mg of wild-type lysine-specific endonuclease (experimental group B) and 50mg of novel lysine-specific endonuclease (experimental group C) were each prepared into a protein solution of 0.5mg/ml using 10mM phosphate buffer, and diluted by 10-fold gradient dilution to obtain a protein solution of 50 ng/ml. The detection is carried out by using a histidine-tag ELISA kit, the light absorption value is detected by using an enzyme-linked immunosorbent assay (ELISAs) instrument at 450nm, 10mM phosphate buffer is taken as a blank control group for the same detection (experiment group A), and the result is shown in figure 3. The results showed that the response values of the wild-type lysine-specific endonuclease (experimental group B) and the blank control group (experimental group A) were equivalent, while the response value of the novel lysine-specific endonuclease (experimental group C) was significant. Therefore, the novel lysine-specific endonuclease can be detected by the histidine-tag ELISA kit, whereas the wild-type lysine-specific endonuclease cannot be detected.
Example 10
Detection of residual quantity of lysine specific endonuclease in preparation of insulin aspart:
dissolving the insulin aspart precursor protein in a solution of 25mM Tris-HCl, 1mM EDTA and pH8.5, adding 2AU wild type lysine specific endonuclease and novel lysine specific endonuclease into each gram of the precursor protein, carrying out enzyme digestion at room temperature (25 ℃) for 24 hours, and analyzing a reaction substrate (insulin aspart precursor protein) and a product (insulin aspart intermediate) in a reaction system by using a High Performance Liquid Chromatograph (HPLC), wherein the result is shown in figure 4. Adding dilute hydrochloric acid into the reaction system to adjust the pH value to 5.0, and standing for 3 hours at 10 ℃. Centrifuging at 8000rpm, 10 deg.C for 10min, collecting precipitate, and vacuum freeze drying to obtain insulin aspart intermediate powder. Respectively carrying out transpeptidation reaction by using wild lysine specific endonuclease and novel lysine specific endonuclease, degreasing, purifying by DEAE ion exchange resin, purifying by C8, and vacuum freeze-drying to obtain insulin aspart powder.
Respectively taking phosphate buffer solution, insulin aspart prepared by using wild lysine specific incision enzyme and insulin aspart prepared by using novel lysine specific incision enzyme. The enzyme residue in the three samples was detected by using His Tag ELISA Detection Kit (as described in the specification) from kasei biotechnology limited, and it was found that the insulin aspart group prepared with the wild-type lysine-specific endonuclease had no response value to the phosphate buffer group, whereas the insulin aspart experimental group prepared with the novel lysine-specific endonuclease had an obvious response value. The residual amount of the novel lysine-specific endonuclease in 1g of insulin aspart was calculated to be 6.2 ng.
The above is only a specific embodiment of the present invention, but the technical features of the present invention are not limited thereto. The technical solutions in the embodiments can also be combined appropriately to form other embodiments that can be understood by those skilled in the art.
SEQUENCE LISTING
<110> Zhuhai Ji Baikang Biotech Co., Ltd
<120> a novel lysyl endonuclease and a method for preparing the same
<130>
<160> 7
<170> PatentIn version 3.5
<210> 1
<211> 274
<212> PRT
<213> Lys-C-1
<400> 1
Gly Val Ser Gly Ser Cys Asn Ile Asp Val Val Cys Pro Glu Gly Asp
1 5 10 15
Gly Arg Arg Asp Ile Ile Arg Ala Val Gly Ala Tyr Ser Lys Ser Gly
20 25 30
Thr Leu Ala Cys Thr Gly Ser Leu Val Asn Asn Thr Ala Asn Asp Arg
35 40 45
Lys Met Tyr Phe Leu Thr Ala His His Cys Gly Met Gly Thr Ala Ser
50 55 60
Thr Ala Ala Ser Ile Val Val Tyr Trp Asn Tyr Gln Asn Ser Thr Cys
65 70 75 80
Arg Ala Pro Asn Thr Pro Ala Ser Gly Ala Asn Gly Asp Gly Ser Met
85 90 95
Ser Gln Thr Gln Ser Gly Ser Thr Val Lys Ala Thr Tyr Ala Thr Ser
100 105 110
Asp Phe Thr Leu Leu Glu Leu Asn Asn Ala Ala Asn Pro Ala Phe Asn
115 120 125
Leu Phe Trp Ala Gly Trp Asp Arg Arg Asp Gln Asn Tyr Pro Gly Ala
130 135 140
Ile Ala Ile His His Pro Asn Val Ala Glu Lys Arg Ile Ser Asn Ser
145 150 155 160
Thr Ser Pro Thr Ser Phe Val Ala Trp Gly Gly Gly Ala Gly Thr Thr
165 170 175
His Leu Asn Val Gln Trp Gln Pro Ser Gly Gly Val Thr Glu Pro Gly
180 185 190
Ser Ser Gly Ser Pro Ile Tyr Ser Pro Glu Lys Arg Val Leu Gly Gln
195 200 205
Leu His Gly Gly Pro Ser Ser Cys Ser Ala Thr Gly Thr Asn Arg Ser
210 215 220
Asp Gln Tyr Gly Arg Val Phe Thr Ser Trp Thr Gly Gly Gly Ala Ala
225 230 235 240
Ala Ser Arg Leu Ser Asp Trp Leu Asp Pro Ala Ser Thr Gly Ala Gln
245 250 255
Phe Ile Asp Gly Leu Asp Ser Gly Gly Gly Thr Pro His His His His
260 265 270
His His
<210> 2
<211> 279
<212> PRT
<213> Lys-C-2
<400> 2
Gly Val Ser Gly Ser Cys Asn Ile Asp Val Val Cys Pro Glu Gly Asp
1 5 10 15
Gly Arg Arg Asp Ile Ile Arg Ala Val Gly Ala Tyr Ser Lys Ser Gly
20 25 30
Thr Leu Ala Cys Thr Gly Ser Leu Val Asn Asn Thr Ala Asn Asp Arg
35 40 45
Lys Met Tyr Phe Leu Thr Ala His His Cys Gly Met Gly Thr Ala Ser
50 55 60
Thr Ala Ala Ser Ile Val Val Tyr Trp Asn Tyr Gln Asn Ser Thr Cys
65 70 75 80
Arg Ala Pro Asn Thr Pro Ala Ser Gly Ala Asn Gly Asp Gly Ser Met
85 90 95
Ser Gln Thr Gln Ser Gly Ser Thr Val Lys Ala Thr Tyr Ala Thr Ser
100 105 110
Asp Phe Thr Leu Leu Glu Leu Asn Asn Ala Ala Asn Pro Ala Phe Asn
115 120 125
Leu Phe Trp Ala Gly Trp Asp Arg Arg Asp Gln Asn Tyr Pro Gly Ala
130 135 140
Ile Ala Ile His His Pro Asn Val Ala Glu Lys Arg Ile Ser Asn Ser
145 150 155 160
Thr Ser Pro Thr Ser Phe Val Ala Trp Gly Gly Gly Ala Gly Thr Thr
165 170 175
His Leu Asn Val Gln Trp Gln Pro Ser Gly Gly Val Thr Glu Pro Gly
180 185 190
Ser Ser Gly Ser Pro Ile Tyr Ser Pro Glu Lys Arg Val Leu Gly Gln
195 200 205
Leu His Gly Gly Pro Ser Ser Cys Ser Ala Thr Gly Thr Asn Arg Ser
210 215 220
Asp Gln Tyr Gly Arg Val Phe Thr Ser Trp Thr Gly Gly Gly Ala Ala
225 230 235 240
Ala Ser Arg Leu Ser Asp Trp Leu Asp Pro Ala Ser Thr Gly Ala Gln
245 250 255
Phe Ile Asp Gly Leu Asp Ser Gly Gly Gly Thr Pro Gly Gly Gly Gly
260 265 270
Ser His His His His His His
275
<210> 3
<211> 284
<212> PRT
<213> Lys-C-4
<400> 3
Gly Val Ser Gly Ser Cys Asn Ile Asp Val Val Cys Pro Glu Gly Asp
1 5 10 15
Gly Arg Arg Asp Ile Ile Arg Ala Val Gly Ala Tyr Ser Lys Ser Gly
20 25 30
Thr Leu Ala Cys Thr Gly Ser Leu Val Asn Asn Thr Ala Asn Asp Arg
35 40 45
Lys Met Tyr Phe Leu Thr Ala His His Cys Gly Met Gly Thr Ala Ser
50 55 60
Thr Ala Ala Ser Ile Val Val Tyr Trp Asn Tyr Gln Asn Ser Thr Cys
65 70 75 80
Arg Ala Pro Asn Thr Pro Ala Ser Gly Ala Asn Gly Asp Gly Ser Met
85 90 95
Ser Gln Thr Gln Ser Gly Ser Thr Val Lys Ala Thr Tyr Ala Thr Ser
100 105 110
Asp Phe Thr Leu Leu Glu Leu Asn Asn Ala Ala Asn Pro Ala Phe Asn
115 120 125
Leu Phe Trp Ala Gly Trp Asp Arg Arg Asp Gln Asn Tyr Pro Gly Ala
130 135 140
Ile Ala Ile His His Pro Asn Val Ala Glu Lys Arg Ile Ser Asn Ser
145 150 155 160
Thr Ser Pro Thr Ser Phe Val Ala Trp Gly Gly Gly Ala Gly Thr Thr
165 170 175
His Leu Asn Val Gln Trp Gln Pro Ser Gly Gly Val Thr Glu Pro Gly
180 185 190
Ser Ser Gly Ser Pro Ile Tyr Ser Pro Glu Lys Arg Val Leu Gly Gln
195 200 205
Leu His Gly Gly Pro Ser Ser Cys Ser Ala Thr Gly Thr Asn Arg Ser
210 215 220
Asp Gln Tyr Gly Arg Val Phe Thr Ser Trp Thr Gly Gly Gly Ala Ala
225 230 235 240
Ala Ser Arg Leu Ser Asp Trp Leu Asp Pro Ala Ser Thr Gly Ala Gln
245 250 255
Phe Ile Asp Gly Leu Asp Ser Gly Gly Gly Thr Pro Gly Gly Gly Gly
260 265 270
Ser Gly Gly Gly Gly Ser His His His His His His
275 280
<210> 4
<211> 289
<212> PRT
<213> Lys-C-4
<400> 4
Gly Val Ser Gly Ser Cys Asn Ile Asp Val Val Cys Pro Glu Gly Asp
1 5 10 15
Gly Arg Arg Asp Ile Ile Arg Ala Val Gly Ala Tyr Ser Lys Ser Gly
20 25 30
Thr Leu Ala Cys Thr Gly Ser Leu Val Asn Asn Thr Ala Asn Asp Arg
35 40 45
Lys Met Tyr Phe Leu Thr Ala His His Cys Gly Met Gly Thr Ala Ser
50 55 60
Thr Ala Ala Ser Ile Val Val Tyr Trp Asn Tyr Gln Asn Ser Thr Cys
65 70 75 80
Arg Ala Pro Asn Thr Pro Ala Ser Gly Ala Asn Gly Asp Gly Ser Met
85 90 95
Ser Gln Thr Gln Ser Gly Ser Thr Val Lys Ala Thr Tyr Ala Thr Ser
100 105 110
Asp Phe Thr Leu Leu Glu Leu Asn Asn Ala Ala Asn Pro Ala Phe Asn
115 120 125
Leu Phe Trp Ala Gly Trp Asp Arg Arg Asp Gln Asn Tyr Pro Gly Ala
130 135 140
Ile Ala Ile His His Pro Asn Val Ala Glu Lys Arg Ile Ser Asn Ser
145 150 155 160
Thr Ser Pro Thr Ser Phe Val Ala Trp Gly Gly Gly Ala Gly Thr Thr
165 170 175
His Leu Asn Val Gln Trp Gln Pro Ser Gly Gly Val Thr Glu Pro Gly
180 185 190
Ser Ser Gly Ser Pro Ile Tyr Ser Pro Glu Lys Arg Val Leu Gly Gln
195 200 205
Leu His Gly Gly Pro Ser Ser Cys Ser Ala Thr Gly Thr Asn Arg Ser
210 215 220
Asp Gln Tyr Gly Arg Val Phe Thr Ser Trp Thr Gly Gly Gly Ala Ala
225 230 235 240
Ala Ser Arg Leu Ser Asp Trp Leu Asp Pro Ala Ser Thr Gly Ala Gln
245 250 255
Phe Ile Asp Gly Leu Asp Ser Gly Gly Gly Thr Pro Gly Gly Gly Gly
260 265 270
Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser His His His His His
275 280 285
His
<210> 5
<211> 286
<212> PRT
<213> Lys-C-5
<400> 5
Gly Val Ser Gly Ser Cys Asn Ile Asp Val Val Cys Pro Glu Gly Asp
1 5 10 15
Gly Arg Arg Asp Ile Ile Arg Ala Val Gly Ala Tyr Ser Lys Ser Gly
20 25 30
Thr Leu Ala Cys Thr Gly Ser Leu Val Asn Asn Thr Ala Asn Asp Arg
35 40 45
Lys Met Tyr Phe Leu Thr Ala His His Cys Gly Met Gly Thr Ala Ser
50 55 60
Thr Ala Ala Ser Ile Val Val Tyr Trp Asn Tyr Gln Asn Ser Thr Cys
65 70 75 80
Arg Ala Pro Asn Thr Pro Ala Ser Gly Ala Asn Gly Asp Gly Ser Met
85 90 95
Ser Gln Thr Gln Ser Gly Ser Thr Val Lys Ala Thr Tyr Ala Thr Ser
100 105 110
Asp Phe Thr Leu Leu Glu Leu Asn Asn Ala Ala Asn Pro Ala Phe Asn
115 120 125
Leu Phe Trp Ala Gly Trp Asp Arg Arg Asp Gln Asn Tyr Pro Gly Ala
130 135 140
Ile Ala Ile His His Pro Asn Val Ala Glu Lys Arg Ile Ser Asn Ser
145 150 155 160
Thr Ser Pro Thr Ser Phe Val Ala Trp Gly Gly Gly Ala Gly Thr Thr
165 170 175
His Leu Asn Val Gln Trp Gln Pro Ser Gly Gly Val Thr Glu Pro Gly
180 185 190
Ser Ser Gly Ser Pro Ile Tyr Ser Pro Glu Lys Arg Val Leu Gly Gln
195 200 205
Leu His Gly Gly Pro Ser Ser Cys Ser Ala Thr Gly Thr Asn Arg Ser
210 215 220
Asp Gln Tyr Gly Arg Val Phe Thr Ser Trp Thr Gly Gly Gly Ala Ala
225 230 235 240
Ala Ser Arg Leu Ser Asp Trp Leu Asp Pro Ala Ser Thr Gly Ala Gln
245 250 255
Phe Ile Asp Gly Leu Asp Ser Gly Gly Gly Thr Pro Gly Gly Ser Gly
260 265 270
Gly Gly Ser Gly Gly Gly Gly Ser His His His His His His
275 280 285
<210> 6
<211> 291
<212> PRT
<213> Lys-C-6
<400> 6
Gly Val Ser Gly Ser Cys Asn Ile Asp Val Val Cys Pro Glu Gly Asp
1 5 10 15
Gly Arg Arg Asp Ile Ile Arg Ala Val Gly Ala Tyr Ser Lys Ser Gly
20 25 30
Thr Leu Ala Cys Thr Gly Ser Leu Val Asn Asn Thr Ala Asn Asp Arg
35 40 45
Lys Met Tyr Phe Leu Thr Ala His His Cys Gly Met Gly Thr Ala Ser
50 55 60
Thr Ala Ala Ser Ile Val Val Tyr Trp Asn Tyr Gln Asn Ser Thr Cys
65 70 75 80
Arg Ala Pro Asn Thr Pro Ala Ser Gly Ala Asn Gly Asp Gly Ser Met
85 90 95
Ser Gln Thr Gln Ser Gly Ser Thr Val Lys Ala Thr Tyr Ala Thr Ser
100 105 110
Asp Phe Thr Leu Leu Glu Leu Asn Asn Ala Ala Asn Pro Ala Phe Asn
115 120 125
Leu Phe Trp Ala Gly Trp Asp Arg Arg Asp Gln Asn Tyr Pro Gly Ala
130 135 140
Ile Ala Ile His His Pro Asn Val Ala Glu Lys Arg Ile Ser Asn Ser
145 150 155 160
Thr Ser Pro Thr Ser Phe Val Ala Trp Gly Gly Gly Ala Gly Thr Thr
165 170 175
His Leu Asn Val Gln Trp Gln Pro Ser Gly Gly Val Thr Glu Pro Gly
180 185 190
Ser Ser Gly Ser Pro Ile Tyr Ser Pro Glu Lys Arg Val Leu Gly Gln
195 200 205
Leu His Gly Gly Pro Ser Ser Cys Ser Ala Thr Gly Thr Asn Arg Ser
210 215 220
Asp Gln Tyr Gly Arg Val Phe Thr Ser Trp Thr Gly Gly Gly Ala Ala
225 230 235 240
Ala Ser Arg Leu Ser Asp Trp Leu Asp Pro Ala Ser Thr Gly Ala Gln
245 250 255
Phe Ile Asp Gly Leu Asp Ser Gly Gly Gly Thr Pro Gly Gly Ser Gly
260 265 270
Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser His His His
275 280 285
His His His
290
<210> 7
<211> 852
<212> DNA
<213> Lys-C-7
<400> 7
ggtgttagcg gcagctgcaa catcgatgtg gtttgcccgg aaggcgatgg tcgtcgtgac 60
atcattcgtg cggtgggtgc gtacagcaaa agcggtaccc tggcgtgcac cggtagcctg 120
gttaacaaca ccgcgaacga ccgtaagatg tattttctga ccgcgcacca ctgcggtatg 180
ggtaccgcga gcaccgcggc gagcattgtg gtttactgga actatcaaaa cagcacctgc 240
cgtgcgccga acaccccggc gagcggtgcg aacggcgatg gtagcatgag ccagacccaa 300
agcggtagca ccgtgaaggc gacctacgcg accagcgact tcaccctgct ggagctgaac 360
aacgcggcga acccggcgtt caacctgttt tgggcgggct gggaccgtcg tgatcagaac 420
tatccgggtg cgatcgcgat tcaccacccg aacgtggcgg aaaaacgtat cagcaacagc 480
accagcccga ccagctttgt tgcgtggggt ggcggtgcgg gtaccaccca cctgaacgtg 540
cagtggcaac cgagcggcgg tgttaccgag ccgggtagca gcggtagccc gatttacagc 600
ccggaaaagc gtgtgctggg tcaactgcac ggcggtccga gcagctgcag cgcgaccggt 660
accaaccgta gcgaccagta tggtcgtgtt ttcaccagct ggaccggcgg tggcgcggcg 720
gcgagccgtc tgagcgactg gctggatccg gcgagcaccg gtgcgcagtt tattgacggt 780
ctggatagcg gtggcggtac cccgggcggt ggcggtagcg gcggcggcgg tagccatcat 840
caccatcacc at 852
Claims (5)
1. A lysine-specific endonuclease, wherein the structure of the endonuclease is as follows: lysine-specific endonuclease core-linker peptide-histidine tag;
the connecting peptide is GGGGSGGGGSGGS;
the histidine tag is 5 consecutive histidines;
the amino acid sequence of the enzyme is shown as SEQ ID NO: 4, respectively.
2. A method for producing the lysine-specific endonuclease according to claim 1, comprising the steps of:
1) constructing a recombinant vector containing the lysine specificity endonuclease coding gene;
2) introducing the recombinant vector into an escherichia coli host cell to obtain a recombinant escherichia coli cell;
3) carrying out fermentation culture on the recombinant escherichia coli cells, adding an inducer, and collecting the expressed recombinant escherichia coli cells;
4) carrying out high-pressure homogenization and crushing on the recombinant escherichia coli cells obtained in the step 3) to obtain a cell suspension containing the novel lysine specific endonuclease;
5) purifying and freeze-drying the cell suspension containing the novel lysine specific endonuclease to obtain lysine specific endonuclease powder.
3. The method according to claim 2, wherein the recombinant vector in step 1) comprises a T7 promoter or a T7lac promoter, a polynucleotide molecule encoding the lysine-specific endonuclease of claim 1, and a T7 terminator located downstream of the polynucleotide molecule, wherein the polynucleotide molecule is located downstream of the promoter.
4. The method of claim 2, wherein the inducing agent in step 3) is 0.3mM IPTG.
5. The method of claim 2, wherein the purification method in step 5) is high pressure homogeneous, nickel column affinity chromatography.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811394022.5A CN109439643B (en) | 2018-11-21 | 2018-11-21 | Novel lysine specific endonuclease and preparation method thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811394022.5A CN109439643B (en) | 2018-11-21 | 2018-11-21 | Novel lysine specific endonuclease and preparation method thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN109439643A CN109439643A (en) | 2019-03-08 |
| CN109439643B true CN109439643B (en) | 2020-12-04 |
Family
ID=65553425
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201811394022.5A Active CN109439643B (en) | 2018-11-21 | 2018-11-21 | Novel lysine specific endonuclease and preparation method thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN109439643B (en) |
Families Citing this family (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109486800B (en) * | 2018-11-21 | 2019-12-13 | 珠海冀百康生物科技有限公司 | Novel lysyl endopeptidase and preparation method thereof |
| CN116334050B (en) * | 2019-11-20 | 2024-02-02 | 珠海联邦制药股份有限公司 | Artificially designed lysyl endonuclease, coding sequence and fermentation method |
| CN115808484B (en) * | 2023-01-18 | 2023-04-14 | 北京惠之衡生物科技有限公司 | Method for detecting residual lysyl endonuclease in insulin or insulin analogue |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101775404A (en) * | 2009-01-08 | 2010-07-14 | 华东理工大学 | Method for highly expressing basic protein with prokaryotic expression system |
| CN105950593A (en) * | 2016-05-13 | 2016-09-21 | 中国人民解放军军事医学科学院放射与辐射医学研究所 | Prokaryotic recombinant expression and preparation method of lysyl endopeptidase |
-
2018
- 2018-11-21 CN CN201811394022.5A patent/CN109439643B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101775404A (en) * | 2009-01-08 | 2010-07-14 | 华东理工大学 | Method for highly expressing basic protein with prokaryotic expression system |
| CN105950593A (en) * | 2016-05-13 | 2016-09-21 | 中国人民解放军军事医学科学院放射与辐射医学研究所 | Prokaryotic recombinant expression and preparation method of lysyl endopeptidase |
Non-Patent Citations (2)
| Title |
|---|
| GenBank登录号AAE57235.1;GenBank;《GenBank登录号》;20010516;参见序列及相关信息 * |
| Lysobacter strain with high lysyl endopeptidase production;Shigeru Chohnan et al;《FEMS Microbiology Letters》;20020716;摘要 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN109439643A (en) | 2019-03-08 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN109486800B (en) | Novel lysyl endopeptidase and preparation method thereof | |
| KR0150565B1 (en) | A process for preparing human proinsulin by recombinant dna technology | |
| CN109439643B (en) | Novel lysine specific endonuclease and preparation method thereof | |
| NO175214B (en) | ||
| CN113105536B (en) | New proinsulin glargine and method for preparing insulin glargine by using same | |
| JPH0822240B2 (en) | Method for removing N-terminal amino acid residues from eukaryotic polypeptide analogs and polypeptides produced thereby | |
| Aiba et al. | Isolation and characterization of the amino and carboxyl proximal fragments of the adenosine cyclic 3', 5'-phosphate receptor protein of Escherichia coli | |
| CN110093336B (en) | Stable trypsin and preparation method thereof | |
| US9611466B2 (en) | Modified enterokinase light chain | |
| KR101527528B1 (en) | Method for production, extraction and purification of soluble recombinant protein | |
| CN103998606B (en) | The Enterpeptidase light chain of modification | |
| CN109136209B (en) | Enterokinase light chain mutant and application thereof | |
| EP3741774A1 (en) | N-terminal fusion partner for producing recombinant polypeptide, and method for producing recombinant polypeptide using same | |
| US8222016B2 (en) | Recombinant C-terminal α-amidating enzyme derivative | |
| CN113061598B (en) | Trypsin mutant, preparation method and application thereof | |
| JP2002529101A (en) | Enzymatic amidation of peptides | |
| KR20190088916A (en) | N-terminal fusion partner for preparing recombinant polypeptide and method of preparing recombinant polypeptide using the same | |
| Chevalier et al. | [31] Purification and properties of BiP | |
| EP1173572B1 (en) | Method of removing n-terminal alanine residues from polypeptides with aeromonas aminopeptidase | |
| KR102009709B1 (en) | Method of preparing human parathyroid hormone 1-84 using fusion polypeptide | |
| JPH0923883A (en) | Method of producing living organism activity beta -ngf by gene control | |
| CN111197041B (en) | Method for preparing medaka enterokinase active subunit, product and application thereof | |
| CN114763552B (en) | Recombinant production method of microbial transglutaminase | |
| JP5114201B2 (en) | Recombinant carboxypeptidase B | |
| Huang et al. | Structure and function of cathepsin D |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |