CN116023472A - Method for producing cysteine protease inhibitor by high-efficiency expression - Google Patents
Method for producing cysteine protease inhibitor by high-efficiency expression Download PDFInfo
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
A method for producing cysteine protease inhibitor by high-efficiency expression comprises the following specific steps: the gene CST7 of the cysteine protease inhibitor is amplified by PCR, and is connected with an expression vector plasmid pPIC9K by double enzyme digestion, recombinant genes are constructed in pichia pastoris, exogenous gene CST7 is introduced, and the gene expression of the CST7 is induced. The engineering bacteria for efficiently expressing the cysteine protease inhibitor constructed by the invention can efficiently produce the cysteine protease inhibitor, and the content of the cysteine protease inhibitor can reach more than 80% after the expressed product is separated and purified. The invention is applied to the field of producing cysteine protease inhibitors.
Description
Technical Field
The present invention relates to a method for preparing a protease inhibitor, and more particularly to a method for producing a cysteine protease inhibitor.
Background
Cysteine protease inhibitors, also known as thiol protease inhibitors, refer to a class of protease inhibitors that reversibly competitively bind to cysteine proteases and have an inhibitory effect on their activity. Distributed in each organism and plays an important role in each physiological and pathological process of the organism. Such as protein metabolism, antigen processing, inflammatory reactions, nutritional disorders and metabolism, and control of enzyme activity. The cysteine protease inhibitor can form a compact complex with acid proteases such as papain, cathepsin and the like, and inhibit the activity of the acid proteases, so that disulfide bonds of proteins are protected from being damaged by the cysteine protease, and the histones are degraded. The plant cysteine protease inhibitor can inhibit the growth of pathogenic fungi such as gibberella, rice blast and the like, and the action mechanism of the plant cysteine protease inhibitor can be related to the inhibition of the invasion of cysteine endopeptidase by the plant cysteine protease inhibitor. Human cysteine protease inhibitors have inhibitory effects on tumor metastasis and viral infection. Cysteine protease inhibitor C is the highest in animal embryos and may have a protective effect on developing embryos. In recent years, it has been reported that cysteine protease inhibitors also play an important regulatory role in apoptosis. Studies on fish cysteine protease inhibitors have shown that cysteine protease inhibitors inhibit the death of fish culture cells caused by baculoviruses. Besides, the method can be used for inhibiting the protease activity of the aquatic products and prolonging the fresh-keeping time in the processing of the aquatic products; cysteine protease inhibitors may also be used in the processing of surimi, and cysteine protease, one of the endogenous proteases responsible for the degradation of surimi gel, catalyzes the hydrolysis of the myosin heavy chain, resulting in the degradation of surimi gel. When the cysteine protease inhibitor is combined with cysteine protease, the degradation of the surimi gel can be effectively inhibited.
Most of the cysteine protease inhibitors currently are derived from natural proteins, of which ovalbumin, whey protein, serum protein and vegetable protein are the four most used proteins. The prior researches show that the plasma protein has higher cysteine protease inhibition activity and is a potential source of natural cysteine protease inhibitors. However, the prevalence of animal diseases such as mad cow disease, avian influenza and the like has long been a serious influence on the raw material sources of the plasma proteins and the safety of the plasma proteins in food processing application. Some cysteine protease inhibitors from fish eggs are severely affected by seasonal factors for fish egg acquisition. Besides the problem of raw material sources, the process for separating and purifying the cysteine protease inhibitor still stays at a small level, the purification steps are complicated, the cost is high, and the subsequent development and use of the inhibitor are seriously affected. If it is desired to obtain a cysteine protease inhibitor without relying on the raw material supply, it can be obtained by using a method of gene expression at the molecular level. According to the existing researches, the most commonly used is the expression of escherichia coli, but most of proteins produced by the escherichia coli are secreted and expressed in the form of inclusion bodies, and cysteine protease inhibitors are obtained after a series of processes of cell disruption, inclusion body disruption, protein renaturation and the like, the activity of the cysteine protease inhibitors is seriously influenced, and most of inhibition activities are lost. The inhibitors thus obtained are also of little utility.
Furthermore, NCBI relates to the cysteine protease inhibitor Gene sequence, NCBI Gene ID:100152012, the present invention performs the subsequent processing based on the sequence.
Disclosure of Invention
The invention provides a method for efficiently expressing and producing a cysteine protease inhibitor, which can efficiently produce the cysteine protease inhibitor; the obtained protein has certain activity and can play an obvious role in inhibiting.
In order to achieve the above object, the present invention provides a method for producing a cysteine protease inhibitor with high expression, comprising the steps of:
s1, designing primers SsCyt/F and SsCyt/R according to a gene sequence of a cysteine protease inhibitor on NCBI, and then performing PCR amplification to obtain a cysteine protease inhibitor CST7;
wherein the Gene sequence NCBI Gene ID of the cysteine protease inhibitor: 100152012;
the primer SsCyt/F sequence is as follows: 5'-atgcgtcctgccagggtgct-3';
the primer SsCyt/R sequence is as follows: 5'-tcagtgacatcggagaacag-3'
S2, carrying out PCR amplification on the inhibitor CST7 by using primers CytF/HF and Cyt/ER, inserting two restriction sites of HindIII and EcoRI, connecting to form a fusion fragment HindIII-CST 7-EcoRI, carrying out double restriction on the fusion fragment by using HindIII and EcoRI, and simultaneously carrying out double restriction on an expression vector pPIC 9K; connecting the fusion fragment of the cysteine protease inhibitor gene CST7 after double digestion with the expression vector pPIC9K after double digestion, and performing heat shock to transfer the fusion fragment into the escherichia coli HB101 strain, and obtaining a recombinant plasmid pPIC9K-CST7 after identification is correct;
wherein, the liquid crystal display device comprises a liquid crystal display device,
the primer CytF/HF sequence is as follows: 5'-cccaagcttatgcgtcctgccagggtgct-3';
the primer Cyt/ER sequence is as follows: 5'-cggaattcgtgacatcggagaacagag-3'
S3, carrying out linearization treatment on the recombinant plasmid pPIC9K-CST7 obtained in the step S2 by using Sac I enzyme, transferring the linearized recombinant plasmid pPIC9K-CST7 into a pichia pastoris (GS 115) strain by electric shock, coating the strain on a RDB plate of a histidine auxotroph medium for screening, transferring the grown bacterial colony onto an MD plate and an MM plate at the same time, and obtaining the bacterial colony which can thrive on both the MD and the MM medium by verifying that the correct positive transformant is the engineering strain;
s4, inoculating the engineering strain obtained in the step S3 into a BMGY culture medium, culturing for 16-24 hours at 230-250rpm and at 28-30 ℃, centrifuging for five minutes at 2000-2500rpm to obtain thalli, and continuing culturing for 72 hours at 230-250rpm and at 28-30 ℃ by using the BMMY culture medium to add methanol into the culture medium every 24 hours until the final concentration is 0.5% -1%; centrifuging the culture medium at 4500-5000rpm for 10min to remove thallus, separating and purifying with nickel ion affinity column to obtain cysteine protease inhibitor.
In a preferred mode, the escherichia coli Strain in the step S2 is selected from the Strain HB101 (HB 101 e.coli Strain) of Shanghai ze biotechnology limited; in step S3, the pichia pastoris strain GS115 produced by the biotechnology limited company of beijing opening research is selected, or the pichia pastoris strain CGMCC2.5688 is selected and purchased from the China general microbiological culture collection center.
The engineering bacteria for efficiently expressing the cysteine protease inhibitor constructed by the invention can efficiently produce the cysteine protease inhibitor, and the content of the cysteine protease inhibitor can reach more than 80% after the expressed product is separated and purified. The invention is applied to the field of producing cysteine protease inhibitors. The invention has the beneficial effects that:
1. according to the invention, a system for expressing the cysteine protease inhibitor is constructed in pichia pastoris, the exogenous gene CST7 is introduced, and the constructed engineering bacteria for efficiently expressing the cysteine protease inhibitor can efficiently produce the cysteine protease inhibitor, so that the purity of the finally purified cysteine protease inhibitor reaches more than 80%.
2. The recombinant plasmid constructed by the invention contains a signal peptide sequence which can transport the recombinant protein outside yeast cells, so that the recombinant protein obtained by expression is convenient for separation and purification.
3. The invention uses yeast cells to express CST7 gene, which can obtain cysteine proteinase inhibitor without being limited by the property of raw materials, seasons and other factors, and the obtained protein has certain activity and can play an obvious role in inhibition. Has great significance for the subsequent research and use and development of cysteine protease inhibitors.
In addition, the protease activity inhibitor secreted by the yeast cell expression system according to the introduced target gene is not secreted and expressed in the form of inclusion bodies, and can be separated and extracted by a protein separation and purification technology, and meanwhile, the protein synthesis of eukaryotes is beneficial to correct folding of the protein and keeps the original activity.
Drawings
FIG. 1 is a nucleic acid electrophoresis diagram of gene CST7.
FIG. 2 is a nucleic acid electrophoretogram of recombinant plasmid pPIC9K-CST7.
FIG. 3 is a nucleic acid electrophoretogram verified by PCR for recombinant yeast transformants.
FIG. 4 is a Tricine-SDS-PAGE of cysteine protease inhibitors.
Detailed Description
The beneficial effects of the invention are verified by the following examples:
example 1: the construction method of the engineering bacteria for producing the cysteine protease inhibitor by high-efficiency expression is carried out according to the following steps:
1. designing primers SsCyt/F and SsCyt/R according to the gene sequence of the cysteine protease inhibitor on NCBI, and then obtaining a cysteine protease inhibitor CST7 by PCR;
2. carrying out PCR amplification on a cysteine protease inhibitor gene CST7 by using primers CytF/HF and Cyt/ER, inserting two restriction sites of HindIII and EcoRI, connecting to form a fusion fragment HindIII-CST 7-EcoRI, carrying out double restriction on the fusion fragment by using HindIII and EcoRI, and simultaneously carrying out double restriction on an expression vector pPIC 9K; connecting the fusion fragment of the cysteine protease inhibitor gene CST7 after double digestion with the expression vector pPIC9K after double digestion, and performing heat shock to transfer the fusion fragment into the escherichia coli HB101 strain, and obtaining a recombinant plasmid pPIC9K-CST7 after identification is correct;
3. linearizing the recombinant plasmid pPIC9K-CST7 obtained in the second step by Sac I enzyme, transferring the linearized recombinant plasmid pPIC9K-CST7 into a pichia pastoris (GS 115) strain by electric shock, coating the strain on a RDB plate of a histidine auxotroph culture medium for screening, transferring the grown bacterial colonies onto MD and MM plates simultaneously, and obtaining positive transformants which are engineering strains after verification, wherein the positive transformants can grow vigorously on the MD and MM culture mediums;
4. and (3) inoculating the engineering strain obtained in the step (III) into a BMGY culture medium, culturing for 16-24h at 230-250rpm and 28-30 ℃, centrifuging for five minutes at 2000-2500rpm, taking thalli, resuspending the thalli with the BMMY culture medium, continuously culturing for 72h at 230-250rpm and 28-30 ℃, and adding methanol into the culture medium every 24h to a final concentration of 0.5% -1%. Centrifuging the culture medium at 4500-5000rpm for 10min to remove thallus, collecting culture medium, and separating and purifying with nickel ion affinity column to obtain cysteine protease inhibitor.
NCBI Gene ID of the cysteine protease inhibitor of step one of this example: 100152012. the primer SsCyt/F sequences in the first step and the second step are as follows: 5'-atgcgtcctgccagggtgct-3'; ssCyt/R sequence is: 5'-tcagtgacatcggagaacag-3'; the primer CytF/HF sequence is as follows: 5' -cccaagcttatgcgtcctgccagggtgct-3'; the Cyt/ER sequence is: 5' -cggaattcgtgacatcggagaacagag-3'。
PCR systems are all
The PCR process is that
The PCR system and the PCR process are the same as those in the first step;
the nucleic acid electrophoresis diagram of the CST7 gene obtained in the first step is shown in FIG. 1, wherein M is DL500 DNAMaroer, and 1 and 2 are genes CST7. As can be seen from FIG. 1, the CST7 gene was successfully amplified.
The nucleic acid electrophoresis diagram of the recombinant plasmid pPIC9K-CST7 obtained in the second step and the double enzyme digestion verification thereof is shown in FIG. 2, wherein M1 is DL15000 DNAMaroer, M2 is DL2000 DNAMaroer, 1 is recombinant plasmid pPIC9K-CST7,2 is HindIII enzyme-digested recombinant plasmid pPIC9K-CST7, and 3 is HindIII enzyme-digested recombinant plasmid pPIC9K-CST7. FIG. 2 shows that the recombinant plasmid pPIC9K-CST7 was constructed successfully.
And C, obtaining a nucleic acid electrophoresis diagram for PCR verification of recombinant yeast transformants, wherein M is DL2000 DNAMaroer, and 1-6 are positive transformants. From FIG. 3, it can be seen that the recombinant yeast was constructed successfully, wherein M was DL2000 DNAMaroer and 1-6 were positive transformants. The constructed engineering bacteria are fermented to produce the cysteine protease inhibitor, which is fermented by taking glycerol as the only carbon source, and is subjected to shaking culture at the temperature of 28-30 ℃ and the speed of 230-250rpm, and the fermentation period is 16-24 hours.
And step four, tricine-SDS-PAGE of the separated and purified cysteine protease inhibitor, wherein M is a low molecular weight Marker,1 is a yeast expression product without introducing CST7 genes, 2 is a sample before separation of a nickel ion affinity chromatographic column, and 3 is a sample after separation of the nickel ion affinity chromatographic column. As can be seen from FIG. 4, the content of the cysteine protease inhibitor can be more than 80%. In FIG. 4, M is a low molecular weight Marker,1 is a yeast expression product into which a CST7 gene is not introduced, 2 is a sample before separation by a nickel ion affinity chromatography column, and 3 is a sample after separation by a nickel ion affinity chromatography column.
The final content of the engineering bacteria for efficiently expressing and producing the cysteine protease inhibitor constructed by the embodiment can reach more than 80 percent.
In this example, pichia pastoris (Pichia pastoris) is a Pichia pastoris CGMCC2.5688 strain, and is purchased from China general microbiological culture collection center.
In the embodiment, recombinant genes for expressing the cysteine protease inhibitor are constructed in pichia pastoris, and exogenous gene CST7 is introduced, so that the constructed cysteine protease inhibitor engineering bacteria can efficiently produce the cysteine protease inhibitor, and the content can reach more than 80 percent finally; the genetically engineered strain obtained by the embodiment can be used for efficiently producing and preparing the cysteine protease inhibitor, so that the preparation cost of the cysteine protease inhibitor is greatly reduced, and the great potential and value of pichia pastoris in the aspect of fermenting and producing the cysteine protease inhibitor are shown, and the genetically engineered strain has great significance for future application of the pichia pastoris.
The foregoing is only a preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art, who is within the scope of the present invention, should be covered by the protection scope of the present invention by making equivalents and modifications to the technical solution and the inventive concept thereof.
Claims (3)
1. A method for producing a cysteine protease inhibitor with high expression comprising the steps of:
s1, designing primers SsCyt/F and SsCyt/R according to a gene sequence of a cysteine protease inhibitor on NCBI, and then performing PCR amplification to obtain a cysteine protease inhibitor CST7;
wherein the gene sequence NCBIGeneID of the cysteine protease inhibitor: 100152012;
the primer SsCyt/F sequence is as follows: 5'-atgcgtcctgccagggtgct-3';
the primer SsCyt/R sequence is as follows: 5'-tcagtgacatcggagaacag-3'
S2, carrying out PCR amplification on the inhibitor CST7 by using primers CytF/HF and Cyt/ER, inserting two cleavage sites of HindIII and EcoRI, connecting to form a fusion fragment HindIII-CST 7-EcoRI, carrying out double cleavage on the fusion fragment by using HindIII and EcoRI, and carrying out double cleavage on an expression vector pPIC 9K; connecting the fusion fragment of the cysteine protease inhibitor gene CST7 after double digestion with the expression vector pPIC9K after double digestion, and performing heat shock and transfer into escherichia coli to obtain a recombinant plasmid pPIC9K-CST7;
wherein, the liquid crystal display device comprises a liquid crystal display device,
the primer CytF/HF sequence is as follows: 5'-cccaagcttatgcgtcctgccagggtgct-3';
the primer Cyt/ER sequence is as follows: 5'-cggaattcgtgacatcggagaacagag-3'
S3, carrying out linearization treatment on the recombinant plasmid pPIC9K-CST7 obtained in the step S2 by using Sac I enzyme, transferring the linearized recombinant plasmid pPIC9K-CST7 into pichia pastoris by electric shock, coating the recombinant plasmid pPIC9K-CST7 on a RDB plate of a histidine auxotroph culture medium for screening, transferring the grown bacterial colonies onto an MD plate and an MM plate at the same time, and obtaining bacterial colonies which can grow on the MD plate and the MM culture medium, namely engineering bacterial strains;
s4, inoculating the engineering strain obtained in the step S3 into a BMGY culture medium, culturing for 16-24 hours at 230-250rpm and at 28-30 ℃, centrifuging for five minutes at 2000-2500rpm to obtain thalli, and continuing culturing for 72 hours at 230-250rpm and at 28-30 ℃ by using the BMMY culture medium to add methanol into the culture medium every 24 hours until the final concentration is 0.5% -1%; centrifuging the culture medium at 4500-5000rpm for 10min to remove thallus, separating and purifying with nickel ion affinity column to obtain cysteine protease inhibitor.
2. The method for producing cysteine protease inhibitors according to claim 1, wherein the E.coli strain in step S2 is selected from the group consisting of Shanghai ze Biotechnology Co., ltd.
3. The method for producing cysteine protease inhibitor according to claim 1, wherein in step S3, pichia pastoris strain GS115 is selected from the group consisting of pichia pastoris strain GS115;
or selecting Pichia pastoris CGMCC2.5688 strain, and purchasing from China general microbiological culture collection center.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101186916A (en) * | 2007-11-20 | 2008-05-28 | 青岛大学 | Gene sequence of coding perinereis albuhitensis grube cysteine protease inhibitor and its amino acid sequence and application |
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| CN114958684A (en) * | 2022-06-22 | 2022-08-30 | 上海龙殷生物科技有限公司 | Method for improving competent cell transformation rate |
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2023
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| WO1997014797A2 (en) * | 1995-10-20 | 1997-04-24 | Dana-Farber Cancer Institute | Cystatin m, a novel cysteine proteinase inhibitor |
| CN101186916A (en) * | 2007-11-20 | 2008-05-28 | 青岛大学 | Gene sequence of coding perinereis albuhitensis grube cysteine protease inhibitor and its amino acid sequence and application |
| CN101423840A (en) * | 2008-12-15 | 2009-05-06 | 大连工业大学 | Method for producing recombinant sea cucumber antalzyme and recombinant sea cucumber antalzyme produced thereby |
| CN114958684A (en) * | 2022-06-22 | 2022-08-30 | 上海龙殷生物科技有限公司 | Method for improving competent cell transformation rate |
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