Preparation method for recombinant protein with optimized expression
By designing and optimizing plasmids, selecting appropriate vectors and DNA assembly technologies, the problem of inclusion bodies formation during the expression of exogenous gene recombinant proteins in bacteria is solved, and efficient soluble expression is achieved.
Patent Information
- Application Number
- CN202411959772.8
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-12-30
- Publication Date
- 2025-05-06
AI Technical Summary
When the exogenous gene recombinant protein is expressed in bacteria, protein aggregation often results in inclusion bodies due to inadequate local microenvironment, and loses biological activity.
By designing plasmids, selecting vector pLysS, gene cloning and DNA synthesis are performed to ensure correct assembly and insertion of DNA elements, combining seamless cloning technology and agarose gel electrophoresis separation, optimize protein expression conditions, and avoid inclusion bodies formation.
It realizes sensitivity and adjustment of exogenous genes during bacterial expression, reduces the protein expression rate, avoids the formation of inclusion bodies, and improves the success rate of soluble expression of recombinant proteins.
Abstract
Description
Technical Field
[0001] The invention relates to the technical field of cell culture, and in particular to a preparation method for optimizing the expression of a recombinant protein. Background Art
[0002] The efficient expression of recombinant proteins from foreign genes in bacteria often causes a considerable amount of protein products to aggregate into inactive solid particles, i.e., inclusion bodies, in the bacteria. The primary structure of these products is completely correct, but the stereo configuration is wrong and they have no biological activity. It is generally believed that the formation of inclusion bodies is caused by the lack of certain auxiliary factors in the expressed foreign proteins or the inappropriate local microenvironment, which prevents the correct and continuous formation of secondary bonds, and the accumulation of intermediate folded bodies at high concentrations.
[0003] To this end, we proposed a preparation method for optimizing recombinant protein expression to solve the problem. Summary of the invention
[0004] 1. Technical issues to be resolved
[0005] In view of the deficiencies in the prior art, the present invention provides a preparation method for optimizing the expression of a recombinant protein, which solves the problems in the above-mentioned background technology.
[0006] (II) Technical solution
[0007] To achieve the above object, the present invention provides the following technical solution: a preparation method for optimizing the expression of a recombinant protein, comprising the following steps: S1: Design plasmid S11: Select carrier: select carrier according to the target; S12: Gene cloning: Determine the sequence of the DNA element to be inserted and construct it using seamless cloning technology; S13: Synthesize or obtain DNA fragments: obtained by artificial synthesis; S14: Plasmid map: Use software to draw a plasmid map to ensure that all components are assembled correctly; S2: Preparation of plasmid and insert S21: Insert fragment: DNA is artificially obtained using the synthetic sequence as a template, and the product is separated and purified by agarose gel electrophoresis; S22: Double digestion: Treat the vector with the selected restriction enzymes to ensure the correct orientation and position; S3: Ligation reaction S31: DNA ligation: Mix the cut vector and insert fragments and add seamless cloning enzyme; S32: Transformation: Transform the ligated plasmid into host cells; S4: Screening clones S41: Plate culture: Spread the transformed cells on agar plates containing chloramphenicol marker; S42: Colony PCR: DNA is manually obtained from the growing colonies to verify the inserted sequence; S43: Colony purification: Select colonies verified by artificially obtained DNA and expand the culture; S5: Plasmid extraction and verification S51: Plasmid extraction: Use a plasmid miniprep or maxiprep kit to extract plasmid DNA from bacteria; S52: Sequence verification: Confirm that the sequence of the inserted fragment is correct by DNA sequencing; S6: Functional Verification S61: Transformation: The above plasmids and expression plasmids were introduced into E. coli cells using the heat shock freezing method; S62: Selection: Add chloramphenicol and other selective antibiotics to select cells carrying the target gene and the above plasmids; S63: Cultivation: Cultivating the selected cells under LB medium culture conditions to promote protein expression; S64: Induced expression: During the culture process, 1mM inducer IPTG was added when OD600 was 0.6-0.8 to activate the promoter and promote the expression of the target protein; S65: Cell disruption: After culturing cells overnight, disrupt the cells to release the target protein; S66: Expression identification: After centrifugation, the supernatant and precipitate samples were taken separately and the expression was identified by SDS-PAGE gel electrophoresis.
[0008] Preferably, the target vector in step S11 is pLysS, which has chloramphenicol resistance and contains a T7 phage lysozyme encoding gene, which strictly inhibits T7 polymerase in the absence of an inducer, reduces the background expression level of the target protein, and is suitable for the expression of toxic proteins or membrane proteins.
[0009] Preferably, the artificial DNA in step S13 can be obtained by chemical synthesis or PCR amplification, wherein chemical synthesis uses chemical reagents to gradually synthesize DNA bases and then connects them into the desired DNA sequence, while PCR amplification uses existing DNA fragments as templates to amplify a large number of target DNA fragments through polymerase chain reaction.
[0010] Preferably, the software in step S14 adopts SnapGene software.
[0011] Preferably, the host cell in step S32 is Escherichia coli.
[0012] Preferably, the cells in step S65 are disrupted by ultrasonic disruption.
[0013] Preferably, the specific method of step S21 is: S211: preparing agarose gel: selecting agarose of appropriate concentration according to needs, heating and dissolving it in a buffer solution and pouring it into a mold to cool and form; S212: Sample loading: Mix the sample containing DNA with the loading buffer and carefully add it to the loading well of the gel; S213: Electrophoresis: When electrophoresis is performed at an appropriate voltage, DNA will separate according to size in the gel; S214: Observation and gel cutting: The position of the DNA bands can be observed by staining, and then the gel part containing the target DNA can be cut out as needed; S215: Extract DNA: Use appropriate methods to extract DNA from the excised gel.
[0014] (III) Beneficial effects
[0015] Compared with the prior art, the present invention provides a preparation method for optimizing the expression of recombinant proteins, which has the following beneficial effects: 1. The present invention has a sensitive perception of the formation of inclusion bodies by exogenous genes during bacterial expression, and spontaneously adjusts the expression rate of exogenous proteins to achieve controllable soluble expression of recombinant proteins.
[0016] 2. The invention has a high success rate in recombinant protein expression, effectively avoiding the cumbersome protein denaturation process, improving efficiency, reducing costs and increasing efficiency. DETAILED DESCRIPTION
[0017] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.
[0018] A method for preparing a recombinant protein by optimizing its expression, characterized in that it comprises the following steps: S1: Design plasmid S11: Select vector: Select pLysS vector according to the target. This vector has chloramphenicol resistance and contains the T7 phage lysozyme encoding gene. It strictly inhibits T7 polymerase in the absence of inducer and reduces the background expression level of the target protein. It is suitable for the expression of toxic proteins or membrane proteins. S12: Gene cloning: Determine the sequence of the DNA element to be inserted and construct it using seamless cloning technology; S13: Synthesize or obtain DNA fragments: obtain through artificial synthesis, specifically by PCR amplification, using existing DNA fragments as templates to amplify a large number of target DNA fragments through polymerase chain reaction; S14: Plasmid map: Use the software SnapGene to draw a plasmid map to ensure that all components are assembled correctly; S2: Preparation of plasmid and insert S21: Insert fragment: DNA is artificially obtained using the synthetic sequence as a template, and the product is separated and purified by agarose gel electrophoresis; The specific method is: S211: preparing agarose gel: selecting agarose of appropriate concentration according to needs, heating and dissolving it in a buffer solution and pouring it into a mold to cool and form; S212: Sample loading: Mix the sample containing DNA with the loading buffer and carefully add it to the loading well of the gel; S213: Electrophoresis: When electrophoresis is performed at an appropriate voltage, DNA will separate according to size in the gel; S214: Observation and gel cutting: The position of DNA bands can be observed by staining (such as ethidium bromide staining), and then the gel part containing the target DNA can be cut out as needed; S215: Extract DNA: Use appropriate methods (such as elution, etc.) to extract DNA from the excised gel; S22: Double digestion: Treat the vector with the selected restriction enzymes to ensure the correct orientation and position; S3: Ligation reaction S31: DNA ligation: Mix the cut vector and insert fragments and add seamless cloning enzyme; S32: transformation: transforming the connected plasmid into a host cell, wherein the host cell is Escherichia coli; S4: Screening clones S41: Plate culture: Spread the transformed cells on agar plates containing chloramphenicol marker; S42: Colony PCR: DNA is manually obtained from the growing colonies to verify the inserted sequence; S43: Colony purification: Select colonies verified by artificially obtained DNA and expand the culture; S5: Plasmid extraction and verification S51: Plasmid extraction: Use a plasmid miniprep or maxiprep kit to extract plasmid DNA from bacteria; S52: Sequence verification: Confirm that the sequence of the inserted fragment is correct by DNA sequencing; S6: Functional Verification S61: Transformation: The above plasmids and expression plasmids were introduced into E. coli cells using the heat shock freezing method; S62: Selection: Add chloramphenicol and other selective antibiotics to select cells carrying the target gene and the above plasmids; S63: Cultivation: Cultivating the selected cells under LB medium culture conditions to promote protein expression; S64: Induced expression: During the culture process, 1mM inducer IPTG was added when OD600 was 0.6-0.8 to activate the promoter and promote the expression of the target protein; S65: Cell disruption: After culturing cells overnight, ultrasonic disruption is used to disrupt the cells and release the target protein; S66: Expression identification: After centrifugation, the supernatant and precipitate samples were taken separately and the expression was identified by SDS-PAGE gel electrophoresis.
[0019] In summary: by introducing two kinds of DNA elements into E. coli, it can sense the "pressure" exerted on exogenous genes during the expression process and actively reduce the expression rate of the protein, eventually tending to reach a steady state or equilibrium state, effectively avoiding the formation of inclusion bodies and achieving effective soluble expression.
[0020] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.
Claims
1. A method for preparing a recombinant protein by optimizing its expression, characterized in that: The following steps are involved: S1: Design plasmid S11: Select carrier: select carrier according to the target; S12: Gene cloning: Determine the sequence of the DNA element to be inserted and construct it using seamless cloning technology; S13: Synthesize or obtain DNA fragments: obtained by artificial synthesis; S14: Plasmid map: Use software to draw a plasmid map to ensure that all components are assembled correctly; S2: Preparation of plasmid and insert S21: Insert fragment: DNA is artificially obtained using the synthetic sequence as a template, and the product is separated and purified by agarose gel electrophoresis; S22: Double digestion: Treat the vector with the selected restriction enzymes to ensure the correct orientation and position; S3: Ligation reaction S31: DNA ligation: Mix the cut vector and insert fragments and add seamless cloning enzyme; S32: Transformation: Transform the ligated plasmid into host cells; S4: Screening clones S41: Plate culture: Spread the transformed cells on agar plates containing chloramphenicol marker; S42: Colony PCR: DNA is manually obtained from the growing colonies to verify the inserted sequence; S43: Colony purification: Select colonies verified by artificially obtained DNA and expand the culture; S5: Plasmid extraction and verification S51: Plasmid extraction: Use a plasmid miniprep or maxiprep kit to extract plasmid DNA from bacteria; S52: Sequence verification: Confirm that the sequence of the inserted fragment is correct by DNA sequencing; S6: Functional Verification S61: Transformation: The above plasmids and expression plasmids were introduced into E. coli cells using the heat shock freezing method; S62: Selection: Add chloramphenicol and other selective antibiotics to select cells carrying the target gene and the above plasmids; S63: Cultivation: Cultivating the screened cells under LB medium culture conditions to promote protein expression; S64: Induced expression: During the culture process, 1mM inducer IPTG was added when OD600 was 0.6-0.8 to activate the promoter and promote the expression of the target protein; S65: Cell disruption: After culturing cells overnight, disrupt the cells to release the target protein; S66: Expression identification: After centrifugation, the supernatant and precipitate samples were taken separately and the expression was identified by SDS-PAGE gel electrophoresis.
2. The method for preparing a recombinant protein with optimized expression according to claim 1, characterized in that: The target vector in step S11 is pLysS, which has chloramphenicol resistance and contains the T7 phage lysozyme encoding gene. It strictly inhibits T7 polymerase in the absence of inducer and reduces the background expression level of the target protein. It is suitable for the expression of toxic proteins or membrane proteins.
3. The method for preparing a recombinant protein with optimized expression according to claim 1, characterized in that: The artificially obtained DNA in step S13 can be obtained by chemical synthesis or PCR amplification. Chemical synthesis uses chemical reagents to gradually synthesize DNA bases and then connects them into the desired DNA sequence, while PCR amplification uses existing DNA fragments as templates to amplify a large number of target DNA fragments through polymerase chain reaction.
4. The method for preparing a recombinant protein with optimized expression according to claim 1, characterized in that: The software used in step S14 is SnapGene software.
5. The method for preparing a recombinant protein with optimized expression according to claim 1, characterized in that: The host cell in step S32 is Escherichia coli.
6. The method for preparing a recombinant protein with optimized expression according to claim 1, characterized in that: The cells in step S65 are disrupted by ultrasonication.
7. The method for preparing a recombinant protein with optimized expression according to claim 1, characterized in that: The specific method of step S21 is: S211: preparing agarose gel: selecting agarose of appropriate concentration according to needs, heating and dissolving it in a buffer solution and pouring it into a mold to cool and form; S212: Sample loading: Mix the sample containing DNA with the loading buffer and carefully add it to the loading well of the gel; S213: Electrophoresis: When electrophoresis is performed at an appropriate voltage, DNA will separate according to size in the gel; S214: Observation and gel cutting: The position of the DNA bands can be observed by staining, and then the gel part containing the target DNA can be cut out as needed; S215: Extract DNA: Use appropriate methods to extract DNA from the excised gel.