CN116064629A - Universal plasmid for escherichia coli surface display system and construction method thereof - Google Patents
Universal plasmid for escherichia coli surface display system and construction method thereof Download PDFInfo
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Abstract
The invention discloses a general surface display system plasmid and a preparation method thereof. According to the invention, INPNC and SpyCatcher form an anchoring fusion protein, target protein and SpyTag form a fusion protein, and the two can still form an isopeptide bond connection at normal temperature. Based on the design, the SpyTag-Linker is connected with the multiple cloning site MCS, and the protein to be displayed can be rapidly inserted through a molecular cloning means, so that the universal surface display system plasmid is prepared.
Description
Technical Field
The invention belongs to the technical field of biology and relates to the technical field of synthetic biology. In particular to a construction method for developing a general plasmid of an escherichia coli surface display system.
Background
The surface display strategy of the bacteria can anchor proteins or polypeptides on the cell outer membrane, and in-situ displayed enzymes can effectively avoid the influence of intracellular factors on enzymatic reaction, and the displayed proteins can be proliferated without secondary manual fixation.
The surface display of bacteria can present protein or polypeptide on the surface of cells, and is widely applied to the fields of whole cell sensors, in-situ catalysts, drug carriers and the like at present. The in-situ enzymatic reaction of the surface display strategy can effectively avoid the influence of some intracellular factors, and meanwhile, the target protein displayed on the surface by the synthetic biology method can be amplified spontaneously along with the bacterial amplification without secondary operation.
However, the current surface display plasmids are still relatively few.
Disclosure of Invention
The main purpose of the invention is to provide a general surface display plasmid and a method for rapidly displaying different proteins of escherichia coli at low cost.
The construction method of the universal plasmid of the escherichia coli surface display system comprises the following steps:
two plasmids J23100-RBS-INPNC-SpyCatcher-Terminator-pSB1C3 and J23100-RBS-HisTag-SpyTag-protein to be expressed-pSB 1C3 were fused by molecular cloning technique to obtain a recombinant plasmid J23100-RBS-INPNC-SpyCatcher-Terminator-J23100-RBS-HisTag-SpyTag-protein to be expressed-pSB 1C 3.
Further, the sequence of J23100-RBS-INPNC-SpyCatcher-Terminator-pSB1C3 is SEQ ID NO:2.
further, J23100-RBS-HisTag-SpyTag-eGFP-pSB1C3 has the sequence SEQ ID NO:3.
further, the resulting recombinant plasmid J23100-RBS-INPNC-SpyCatcher-Terminator-J23100-RBS-HisTag-SpyTag-protein to be expressed-pSB 1C3, after removal of eGFP, has the sequence SEQ ID NO:4.
further, the method comprises the following steps: the CDS sequence of the protein to be displayed is amplified by a forward primer and a reverse primer with Sal I and BamH I enzyme cutting sites respectively, and a PCR product and an Easy-Anchoring sequence are subjected to double enzyme cutting by using restriction enzymes Sal I and BamH I to obtain a fragment to be inserted and an expression skeleton; finally, the fragment was ligated using T4 ligase to form a plasmid.
The ice-nucleating protein (INP) comprises an N-terminal domain, a C-terminal structure and a central repeat domain. The C-terminal and central repeat domains are not necessary for anchoring functions. Thus, when only the N-terminal region is applied to the E.coli surface, the surface display of the enzyme can be achieved. Meanwhile, the ice nuclear protein has secretion, guidance and anchoring functions, and the fusion protein can be self-anchored on the cell surface without other specific auxiliary proteins.
The pSB1C3 plasmid is a chloramphenicol resistant plasmid applicable to E.coli, and the backbone does not contain the four common restriction sites EcoR I, xba I, spe I, pst I, so that plasmid recombination can be performed using the standard prefix and suffix containing these four sites using the RFC 10 assembly rule.
The ice nucleoprotein (INPNC) is an anchored protein from Pseudomonas syringae. It can anchor the target protein to the cell membrane. The N-and C-termini of ice nucleoproteins (named INPNC) can also anchor proteins fused thereto to the cell membrane. SpyTag is a peptide fragment that can be stably bound to SpyCatcher by formation of an isopeptide bond. The multiple cloning site MCS is an artificially synthesized DNA fragment contained on a vector, which contains a plurality of single cleavage sites, into which exogenous DNA fragments can be inserted. INPNC and SpyCatcher form an anchoring fusion protein, target protein and SpyTag form a fusion protein, and the two can still form isopeptide bond connection at normal temperature. Based on the design, the SpyTag-Linker is connected with the multiple cloning site MCS, and the protein to be displayed can be rapidly inserted through a molecular cloning means.
Compared with the prior art, the invention has the beneficial effects that:
1. according to the invention, the anchoring of different proteins can be realized by only changing the eGFP sequence of the J23100-RBS-HisTag-SpyTag-eGFP-pSB1C3 without changing the sequence of the plasmid J23100-RBS-INPNC-SpyCatcher-Terminator-pSB3K3, and the operation is convenient.
2. The surface display method can directly self-assemble and fix the protein without special reagents, and is green, economical and environment-friendly.
3. The surface display system can express the protein without induction, and is simple in operation, convenient and efficient.
Drawings
The invention is further described below with reference to the drawings and examples.
FIG. 1 is a schematic diagram of the use of a generic plasmid.
FIG. 2 recombinant plasmid nucleic acid gel diagram: DNA gel electrophoresis of EcoR l & PstI site restriction digest;
FIG. 3 eGFP surface shows TEV cleavage fluorescence/OD 600 comparison results
FIG. 4 fluorescence confocal microscopy Dil membrane dye co-localizes INPNC protein with eGFP protein, scale bar: 2. Mu.m.
Detailed Description
The present invention uses the model protein eGFP as an example, and aims to verify the function of the recombinant plasmid. Two plasmids J23100-RBS-INPNC-SpyCatcher-Terminator-pSB1C3 and J23100-RBS-HisTag-SpyTag-eGFP-pSB1C3 were fused by molecular cloning techniques to obtain a recombinant plasmid J23100-RBS-INPNC-SpyCatcher-Terminator-J23100-RBS-HisTag-SpyTag-eGFP-pSB1C 3. The fusion plasmids are respectively transferred into escherichia coli BL21 (DE 3) to construct engineering bacteria with the surface displaying eGFP protein.
The invention designs a multiple cloning site based on the gene, and can conveniently and directly enter other target protein sequences. The multiple cloning site contains only Sal I and BamH I, and the fusion plasmid Easy-fastening containing the multiple cloning site J23100-RBS-INPNC-SpyCatcher-Terminator-J23100-RBS-HisTag-SpyTag-MCS-pSB1C3 (MCS) can be introduced into the novel CDS by molecular cloning means. The method is as shown in fig. 1:
the CDS sequence of the protein to be displayed is amplified by a forward primer and a reverse primer with Sal I and BamH I enzyme cutting sites respectively, and the general partial sequence of the primer is as follows: (F: ACGCGTCGAC, R: CGCGGATCC). Both the PCR product and Easy-fastening sequence (SEQ ID NO: 4) were digested with the restriction enzymes Sal I and BamH I to give the desired insert and the expression cassette. And finally, using T4 ligase to carry out fragment connection and converting the fragment connection into escherichia coli BL21 (DE 3), thus constructing the surface display engineering bacteria.
Specifically, the technical scheme of the invention is as follows:
(1) Construction of J23100-RBS-INPNC-SpyCatcher-Terminator-J23100-RBS-HisTag-SpyTag-eGFP-pSB1C3 vector
J23100-RBS-INPNC-SpyCatcher-Terminator sequence of 1481bp in length and J23100-RBS-HisTag-SpyTag-eGFP sequence of 938bp in length (both on pSB1C3 vector) were designed, and a prefix satisfying the RFC 10 assembly rule was added before and after the sequence: contains four restriction sites of EcoR I, xba I, spe I and Pst I.
The two sequences were obtained from laboratory molecular cloning. Then the J23100-RBS-HisTag-SpyTag-eGFP-pSB1C3 plasmid is cut off and inserted between the cutting sites of the SpeI and the Pst I of the J23100-RBS-INPNC-Spycatcher-Terminator-pSB1C3 vector by molecular cloning. The assembled DNA sequence (J23100-RBS-INPNC-SpyCatcher-J23100-RBS-HisTag-SpyTag-eGFP) is shown in SEQ ID NO: 1. The final recombinant plasmid nucleic acid validation gel results are shown in FIG. 2. The Easy-Anchoring sequence designed based on the method is shown as SEQ ID NO:4.
(2) Transformation and expression
Adding 50 mu L of competent cells into a 1.5mL centrifuge tube, adding 5 mu L of plasmid to be transferred, and blowing and mixing uniformly; immediately after incubation on ice for 30min, an ice bath was made for 2min after heat shock at 42℃for 45 s. To the centrifuge tube, 450. Mu.L of LB medium was added and incubated for 1.5h at 37℃in a shaker at 200 rpm. mu.L of the culture was plated on LB solid medium and cultured overnight at 37 ℃. And (5) extracting bacterial liquid plasmid sequencing to verify correctness.
And inoculating the monoclonal colony with correct sequence into LB culture medium containing antibiotics, and culturing at 37 ℃ at 200rpm for 16h to express the target protein.
(3) TEV enzyme digestion verification
TEV enzyme is a cysteine protease from tobacco etch virus (Tobacco Etch Virus, TEV) that specifically recognizes the heptapeptide sequence Glu-Asn-Leu-Tyr-Phe-Gln-Gly/Ser for cleavage. The reaction is carried out by using a Beyotime P2307 TEV Protease (His-tag) kit: bacteria required for the reaction were cultured overnight, centrifuged at 6500rpm and washed with PBS for resuspension. And centrifuging again to obtain clean thalli. 1000. Mu.L of PBS was added to the cells, and OD600 was measured for use. Wherein the configuration of the reaction system: 43.5. Mu.L of bacterial liquid, 1.5. Mu.L of TEV enzyme and 2.5. Mu.L of buffer. The PCR was performed at 30℃for 1.5 hours. Take out 9000rpm and centrifuge for 2min, take out supernatant for downstream reaction.
The sequence was designed by inserting TEV Site between INPNC and Spycatcher, in contrast to E.coli which directly expressed eGFP. And taking supernatant obtained by experiments after TEV enzyme digestion reaction, and carrying out fluorescence measurement. The results show that the PBS group is non-fluorescent and the surface display eGFP group has higher fluorescence/OD 600 value than the direct expression eGFP group. Experiments prove that eGFP is successfully displayed. The results are shown in FIG. 3.
(4) Membrane protein localization experiments
Confocal Dil membrane dyes were used to localize the surface displayed eGFP proteins. DiI is one of the most commonly used cell membrane fluorescent probes, exhibiting orange-red fluorescence. DiI is a lipophilic membrane dye that fluoresces very weakly before entering the cell membrane and can be excited very strongly only after entering the cell membrane. Leica TCS SP 8X super-resolved fluorescence confocal microscopy experimental conditions: surface display eGFP-488 nm excitation, 471-517nm emission; diI localization-552 nm excitation, 552-585nm emission. The DiI-localized outer membrane was observed to overlap with the outer membrane-displayed eGFP fluorescence, indicating that the surface display system is functioning properly.
Sequence1 (with prefix and suffix, without pSB1C3 backbone)
Sequence2
Sequence3
Sequence4 (containing pSB1C3 backbone, labeled with MCS Sequence)
The underlined part is the multiple cloning site for insertion of the protein to be displayed.
Claims (9)
1. A general plasmid of an escherichia coli surface display system, which removes a protein gene to be displayed, wherein the sequence of the protein gene is shown as SEQ ID NO:4.
2. The construction method of the universal plasmid of the escherichia coli surface display system comprises the following steps:
two plasmids J23100-RBS-INPNC-SpyCatcher-Terminator-pSB1C3 and J23100-RBS-HisTag-SpyTag-protein to be expressed-pSB 1C3 were fused by molecular cloning technique to obtain a recombinant plasmid J23100-RBS-INPNC-SpyCatcher-Terminator-J23100-RBS-HisTag-SpyTag-protein to be expressed-pSB 1C 3.
3. The method for constructing an escherichia coli surface display system according to claim 2, wherein the method comprises the following steps: J23100-RBS-INPNC-SpyCatcher-Terminator-pSB1C3 has the sequence of SEQ ID NO:2.
4. the method for constructing a universal plasmid for an escherichia coli surface display system according to claim 2, wherein the method comprises the following steps: J23100-RBS-HisTag-SpyTag-eGFP-pSB1C3 the sequence after removal of eGFP is SEQ ID NO:3.
5. the method for constructing a universal plasmid for an escherichia coli surface display system according to claim 2, wherein the method comprises the following steps: the resulting recombinant plasmid J23100-RBS-INPNC-SpyCatcher-Terminator-J23100-RBS-HisTag-SpyTag-protein to be expressed-pSB 1C3, after eGFP removal, has the sequence SEQ ID NO:4.
6. the method for constructing a universal plasmid for an escherichia coli surface display system according to claim 2, wherein the method comprises the following steps: the method comprises the following steps:
the CDS sequence of the protein to be displayed is amplified by a forward primer and a reverse primer with Sal I and BamH I enzyme cutting sites respectively, and a PCR product and an Easy-Anchoring sequence are subjected to double enzyme cutting by using restriction enzymes Sal I and BamH I to obtain a fragment to be inserted and an expression skeleton; finally, the fragment was ligated using T4 ligase to form a plasmid.
7. The method of claim 6, wherein the plasmid is further transformed into E.coli to construct a surface display engineering bacterium.
8. The method of claim 7, wherein the E.coli strain is E.coli BL21 (DE 3).
9. The method for constructing a universal plasmid for an escherichia coli surface display system according to claim 2, wherein the method comprises the following steps: the method comprises the following steps:
construction of J23100-RBS-INPNC-SpyCatcher-Terminator-J23100-RBS-HisTag-SpyTag-eGFP-pSB1C3 vector
J23100-RBS-INPNC-SpyCatcher-Terminator sequence of 1481bp in length and J23100-RBS-HisTag-SpyTag-eGFP sequence of 938bp in length were designed: four restriction sites containing EcoR I, xba I, spe I and Pst I;
then cutting the J23100-RBS-HisTag-SpyTag-eGFP-pSB1C3 plasmid by a molecular cloning means, and inserting the J23100-RBS-HisTag-SpyTag-eGFP fragment between the Spe I and the Pst I cleavage sites of the J23100-RBS-INPNC-Spycatcher-Terminator-pSB1C3 vector; the assembled DNA sequence J23100-RBS-INPNC-SpyCatcher-J23100-RBS-HisTag-SpyTag-eGFP is shown in SEQ ID NO: 1.
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| CN108486024A (en) * | 2018-03-20 | 2018-09-04 | 上海交通大学 | The method of sensor-based system detection organophosphorus pesticide based on flora |
| CN108676808A (en) * | 2018-05-25 | 2018-10-19 | 厦门大学 | A kind of recombinant plasmid pET28a-Ag43, surface showing plasmid, recombination engineering and its application |
| CN110669132A (en) * | 2019-08-19 | 2020-01-10 | 天津大学前沿技术研究院 | Method for improving IgG1 antibody yield |
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