CN112342211A - Protein interaction detection method based on split-HRV 3C protease - Google Patents
Protein interaction detection method based on split-HRV 3C protease Download PDFInfo
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Abstract
The invention relates to a protein interaction detection method based on a split-HRV 3C protease-E.coli ClpXP system, which has the following principle: due to the presence of the ClpXP-SsrA degradation pathway, the fusion protein fusing sfGFP-HRV 3C protease substrate polypeptide-SsrA can be recognized by an Escherichia coli ClpXP complex, so that the sfGFP is degraded and low-intensity green fluorescence is presented. The capture protein and the bait protein are respectively fused in the N-terminal domain and the C-terminal domain of split-HRV 3C protease, when the capture protein and the bait protein interact, the HRV 3C protease at the N terminal and the C terminal is fused to form the HRV 3C protease with complete function, and the corresponding substrate polypeptide is cut and identified, so that sfGFP accumulation and fluorescence intensity are improved. According to the invention, by integrating the ClpXP-SsrA degradation machine and sfGFP and split-HRV 3C protease, the protein-protein interaction can be efficiently and sensitively detected in a wide temperature range according to the green fluorescence intensity.
Description
Technical Field
The invention relates to the field of protein interaction research, in particular to a protein interaction detection method based on split-HRV 3C protease.
Background
The study of protein interactions is of vital biological importance for understanding metabolic processes in the body and the functions of the proteins involved. Today, many methods for studying protein interactions have been developed, such as: bacteria/yeast two-hybrid technology, Co-immunoprecipitation technology (Co-IP), GST pull down technology, split-protein complementation technology, and the like. The yeast two-hybrid technology is characterized by sensitivity, high efficiency, simple operation and large-scale verification, and in practical application, the yeast two-hybrid technology is accompanied by certain false positive due to the sensitivity of yeast two-hybrid, and the reaction is generated in cell nucleus, so the yeast two-hybrid technology is not suitable for all proteins. Meanwhile, the Co-IP uses an immune label to have false positive interaction, GST pull down needs to express purified protein in vitro, and certain limitation exists.
Disclosure of Invention
The invention aims to provide a protein interaction detection method utilizing split-HRV 3C protease based on an Escherichia coli ClpXP degradation machine (SPEC) aiming at the problems.
The technical scheme for solving the technical problems is as follows:
the protein interaction detection method based on the split-HRV 3C protease comprises the following steps:
and 4, transferring the first recombinant protein and the second recombinant protein into an escherichia coli recombinant strain for co-expression, detecting a green fluorescent signal of the recombinant escherichia coli cell by using a flow cytometer, if the green fluorescent signal is detected, indicating that interaction exists between the capture protein and the bait protein, and if no green fluorescent signal is detected, indicating that interaction does not exist between the capture protein and the bait protein.
Further, the cleavage site of the HRV 3C protease in the step 2 is K82 site.
Further, the SsrA degradation tag can be recognized by the endogenous ClpXP degradation complex of E.coli, resulting in degradation of the SsrA N-terminal fused protein.
Furthermore, an N end of the HRV 3C protease substrate is fused with sfGFP, and a C end is fused with an SsrA degradation label; the HRV 3C protease substrate polypeptide is sfGFP-LEVLFQGP-SsrA.
Further, the capture protein and the bait protein are a target protein pair of interaction to be researched; the capture protein is fused at the N-terminal HRV 3C protease domain, and the bait protein is fused at the C-terminal HRV 3C protease domain.
Further, when the capture protein and the bait protein interact, the HRV 3C protease at the N end and the C end is fused to form the HRV 3C protease with complete function, and the cleavage and recognition of corresponding substrate polypeptide are carried out, so that the accumulation of sfGFP signals and the increase of the fluorescence intensity of cells are caused.
According to the method for detecting the protein interaction based on the split-HRV 3C protease, the sfGFP signal can be detected by a flow cytometer.
An application of a split-HRV 3C protease-E.coli ClpXP system in detecting whether proteins interact and the interaction strength is disclosed, wherein the split-HRV 3C protease-E.coli ClpXP system combines the functions of a ClpXP-SsrA degradation machine, sfGFP and split-HRV 3C protease.
A preparation method of an escherichia coli recombinant strain comprises the following steps: step 1, fusing superfolder green fluorescent protein sfGFP at the N end of HRV 3C protease substrate sequence LEVLFQ ↓ GP, and fusing SsrA degradation label at the C end of HRV 3C protease substrate sequence to obtain HRV 3C protease substrate polypeptide sfGFP-LEVLFQGP-SsrA;
use of recombinant E.coli strains prepared by the method of claim for detecting protein interactions and interaction strength
The invention has the beneficial effects that:
1. the SPEC system carries out secondary cascade amplification on a protease hydrolysis signal and a green fluorescence signal through the integration of a ClpXP-SsrA degradation machine and sfGFP and split-HRV 3C protease, so that the sensitivity is improved;
2. the HRV 3C protease selected by the SPEC system has stronger activity, and when the HRV 3C protease works in the SPEC system, the activity of the HRV 3C protease is four times that of TEV protease, so that better sensitivity can be exerted; and has stronger temperature application range (18-37 ℃), and has good activity at 18-37 ℃;
3. the SPEC system of the present invention uses a flow cytometer for detecting protein-protein interactions and E.coli as the host of operation, which is simple to operate.
Drawings
FIG. 1 is a schematic diagram of the process of the present invention;
FIG. 2 is a schematic structural diagram of HRV 3C protease;
FIG. 3 is a plasmid map of vectors SPEC-VA, SPEC-VB, SPEC-VD, SPEC-VE;
FIG. 4 shows the results of comparative tests of HRV 3C protease and TEV protease at different temperature ranges (37,30,25 and 18 ℃);
FIG. 5 shows the flow detection results of split-HRV 3C protease at three different split positions (k82, L94, N107) in BL21(DE3) for the target protein pairs of Cas1/Cas2-3 derived from Zymomonas mobilis and Yae1/Lto1 derived from Saccharomyces cerevisiae, respectively;
FIG. 6 shows the results of comparative tests on split-HRV 3C protease and split-TEV protease in BL21(DE3), respectively, at different temperature ranges (37,30,25 and 18 ℃) for a target protein pair Cas1/Cas2-3 derived from Zymomonas mobilis;
FIG. 7 shows the results of comparative tests of split-HRV 3C protease and split-TEV protease in BL21(DE3), respectively, at different temperature ranges (37,30,25 and 18 ℃) for the target protein pair Yae1/Lto1 from Saccharomyces cerevisiae;
FIG. 8 is a flowchart showing the integration of the sfGFP-LEVLFQGP-SsrA fragment into the chromosome of E.coli and the verification of the recombinant strain BL21(DE3) -SPEC;
FIG. 9 shows the results of comparative tests of split-HRV 3C protease in recombinant strain BL21(DE3) -SPEC at different temperatures (37,30,25 and 18 ℃) for the target protein pairs Zymomonas mobilis Cas1/Cas2-3 and Saccharomyces cerevisiae Yae1/Lto1, respectively;
Detailed Description
The principles and features of this invention are described below in conjunction with the following drawings, which are set forth by way of illustration only and are not intended to limit the scope of the invention.
The invention establishes a novel technology for researching protein-protein interaction, namely split HRV 3C protease-E.coli ClpXP (SPEC, a split-HRV 3C protease system based on an escherichia coli ClpXP degradation machine). As shown in FIG. 1, first, sfGFP (super-fold green fluorescence protein) was fused to the N-terminus of HRV 3C protease substrate sequence (LEVLFQ ↓ GP), and SsrA degradation tag sequence (AANDENYALAA) was fused to the C-terminus of HRV 3C protease substrate sequence, forming a sfGFP-LEVLFQ ↓ GP-SsrA cassette structure. Meanwhile, HRV 3C protease (Human rhinovirus 3C protease) and TEV protease (tobacoetch virus protease) are both virus-derived proteases having high proteolytic activity and strong substrate specificity, and thus are good candidates for SPEC system proteases. HRV 3C protease has faster cleavage ability and at low temperature range compared to TEV protease(4 ℃ to 37 ℃) ability to maintain high activity, and is referred to as Raran-Kurussi, S.,
J.,Cherry,S.,Tropea,J.E.,and Waugh,D.S.Differential temperature dependence of tobacco etch virus and rhinovirus 3C proteases.Anal.Biochem.，2013；436,142-144]. The HRV 3C protease structure comprises two subdomains of a six-stranded β -barrel structure joined by random helices, referenced in Fan, x.et al.quantitative analysis of the substrate specificity of human rhenovirus 3C protease and expression of its substrate recognition mechanisms acs biol, 2019; 15,63-73]. As shown in fig. 2, the three residues K82, L94 and N107 in the HRV 3C protease structure are located on their random helices and subsequent β -pleated sheet domains, which are used to generate the split-HRV 3C protease. As shown in FIG. 1, the prey protein and the bait protein were then fused to the N-and C-termini, respectively, of the split-HRV 3C protease. In principle, sfGFP-LEVLFQ ↓ GP-SsrA highly expressed in E.coli is recognized by the ClpXP degradation complex due to the presence of the endogenous ClpXP-SsrA degradation pathway of E.coli, resulting in the degradation of sfGFP, thereby presenting a very low green fluorescence background (FIG. 1). Nevertheless, when the capture protein (prey protein) and the bait protein (Bait protein) interact, the two domains of the split-HRV 3C protease at the N-terminal and the C-terminal are fused again to form an HRV 3C protease with complete function, and the cleavage recognition of sfGFP-LEVLFQ ↓GP-SsrA is carried out, so that the SsrA degradation tag is dropped, and the accumulation of sfGFP and the increase of fluorescence intensity are caused.
Example 1
Construction of vectors SPEC-VA and SPEC-VB for expressing HRV 3C protease substrate polypeptide and wild-type HRV 3C protease, respectively, and comparison of working effects of wild-type HRV 3C protease and wild-type TEV protease
(1) Firstly, two target genes of sfGFP-LEVLFQGP-SsrA and HRV 3C protease are obtained by Polymerase Chain Reaction (PCR) amplification, and the reaction system is as follows: 50 μ L system, 10 XKOD buffer, 5 μ L; dNTP (2.5mM), 3. mu.L; forward primer (10 μ M), 2 μ L; reverse primer (10. mu.M), 2. mu.L; pfu high temperature DNA polymerase, 1. mu.L; template (containing sfGFP-LEVLFQGP-SsrA sequence), 0.5. mu.L (20 ng/. mu.L); add double distilled water to 50. mu.L.
PCR amplification conditions: 95 ℃ for 5 min; 25 cycles of 95 ℃,30 s, 57 ℃,30 s, 72 ℃,30 s; 72 ℃ for 5 min; 12 ℃ for 10 min.
The primers were designed as follows:
T7-FP:GCCTGGTGCCGCGCGGCAGCCATATGATGGTGAGCAAGGGCGAG
T7-RP:CTTGTCGACGGAGCTCGAATTCGGATCCGGCTGCCAAAGCATAGTTTTC
1928-HRV3C-Fwd:GTGCCGCGCGGCAGCCATATGGCTAGCGGTCCAAATACTGAATTTGC
1928-HRV3C-Rvs:CTTGTCGACGGAGCTCGAATTCGGATCCTCAAGAACCTTGCTTTTCAAC
(2) recovering the target fragment by agarose gel, performing double digestion with NdeI and BamHI at 37 ℃ for 5hr, and recovering by agarose gel, wherein the double digestion system comprises: target fragment DNA, 30. mu.L; 10 × Cutsmart Buffer, 5 μ L; 2 μ L of each of NdeI and BamHI; add double distilled water to 50. mu.L.
(3) Respectively carrying out enzyme digestion on the vector p1928 by the same system and recovering, carrying out enzyme linked vector and the target fragment at 22 ℃ for 2h, carrying out enzyme linked reaction: vector, 1.2 μ L; target fragment, 0.5 μ L; t4 DNA Ligase, 0.2. mu.L (thermo Fisher), 10 XT 4 Ligase Buffer, 2. mu.L; double distilled water was added to 20. mu.L.
(4) And (3) transforming, taking 5 mu L of the enzyme-linked product, placing the enzyme-linked product in 50 mu L of XL-glod competence, standing the product on ice for 10min, then carrying out heat shock at 42 ℃ for 45s, adding LB culture medium, carrying out shake culture at 37 ℃ and 250rpm for 1h, and then coating an Amp resistant plate.
(5) Sequencing 2 recombinants identified by colony PCR were selected each to obtain correctly recombined HRV 3C protease substrate expression vector SPEC-VA and wild-type HRV 3C protease expression vector SPEC-VB (as shown in FIG. 3).
(6) Vectors for expressing wild-type HRV 3C protease and wild-type TEV protease and corresponding substrate expression vectors were co-transformed into E.coli BL21(DE3) expression hosts, spread on LB double antibody plates containing kanamycin (final concentration 100. mu.g/mL) and ampicillin (50. mu.g/mL), and cultured in 37 ℃ incubator for 12-16 hours. The LB solid medium comprises 5g/L yeast extract, 10g/L peptone, 10g/L sodium chloride and 15g/L agar.
A single colony was inoculated in liquid LB medium containing kanamycin (final concentration 100. mu.g/mL) and ampicillin (50. mu.g/mL). After incubation at 37 ℃ in a shaker at 220rpm to an OD600 of 0.6, induction was carried out at different temperatures (37,30,25 and 18 ℃) and a final concentration of 0.5mM IPTG, samples were taken at intervals (100. mu.L/time), the supernatant was centrifuged off, washed twice with 150. mu.L of 1 XPBS and resuspended in 400. mu.L. The resuspended cells are used for cytoFLEX flow cytometry analysis, the detected fluorescence signal channel is FITC (BP 525/40nm), the average fluorescence intensity of sample cells is measured, and compared with the working effect of detecting wild-type HRV 3C protease and wild-type TEV protease at different temperatures (37,30,25 and 18 ℃), as shown in figure 4, the result shows that the HRV 3C protease activity is nearly 4 times of that of the TEV protease, the HRV 3C protease can show good activity at 18-37 ℃, and the highest green fluorescent protein expression level is shown under the induction of IPTG (4, 6,10 and 20h respectively); however, TEV protease showed a tendency to decrease in activity at 18-30 ℃ indicating that HRV 3C protease has a broader temperature range adaptability than TEV protease.
Example 2
Construction process of split-HRV 3C protease carrier with different split positions and comparison of working effects of split-HRV 3C protease and split-TEV protease in different temperature ranges
Analyzing the three-dimensional structure of the HRV 3C protease, finding out three possible split sites (K92, L94, N107) which are respectively positioned behind a random spiral sheet and a beta-folded sheet, and then respectively constructing split-HRV 3C protease vectors at three split positions of K92, L94 and N107 according to the vector construction mode in example 1; the selected target protein pairs are Cas1/Cas2-3 derived from Zymomonas mobilis and Yae1/Lto1 derived from Saccharomyces cerevisiae.
The primers were designed as follows:
NC-FP1:GATTACGCCAAGCTTGCATGCCTGCAGTTGACAATTAATCATCGGCTCG
NC-RP1:CATACATCGTCCCAAGTATTCGACATGAATTCAATCTATGGTCCTTG
NC-FP2:CAAGGACCATAGATTGAATTCATGTCGAATACTTGGGACGATGTATGGGC
NC-RP2:GCTCCCGCCGCCACCACTACCACCGCCTCCTTGGATAGATGGCACATGG
NC-FP3:GTAGTGGTGGCGGCGGGAGCGGTCCAAATACTGAATTTGC
NC-RP3:CGTTTTATTTGAGATCTTCACTTTTCATTGCGATCCAAAGTCAATAC
NC-FP4:GTGAAGATCTCAAATAAAACGAAAGGCTCAGTCGAAAGACTG
NC-RP4:GTAAAACGACGGCCAGTGAATTCGAGCTCAAGAGTTTGTAGAAACGC
NC-FP5:GATTACGCCAAGCTTGCATGCCTGCAGTTGACAATTAATCATCGGCTCG
NC-RP5:GTAAATTATCAAAATCCTCGAGAATCTATGGTCC
NC-FP6:GACCATAGATTCTCGAGATGGATTTTGATAATTTACTAAACC
NC-RP6:GCTCCCGCCGCCACCACTACCACCGCCTCCCCAGGATTGAGCCTGATTTTG
NC-FP7:GGTAGTGGTGGCGGCGGGAGCTTTCGTGATATTCGTGGTTTC
NC-RP7:GAGCCTTTCGTTTTATTTGGGATCCTCAAGAACCTTGCTTTTC
NC-FP8:CAAATAAAACGAAAGGCTCAGTCGAAAGACTGGGC
NC-RP8:GTAAAACGACGGCCAGTGAATTCGAGCTCAAGAGTTTGTAGAAACGC
CAS1-F:CACCAACAAGGACCATAGATTGAATTCATGAAGCCAATAACATCGG
CAS1-R:GCTCCCGCCGCCACCACTACCACCGCCTCCTCATAATGCAGGCTCGGCCTCC
CAS2-F:CAAGGACCATAGATTCTCGAGATGAGTATGTTGGTCGTGG
CAS2-R:GCTCCCGCCGCCACCACTACCACCGCCTCCTCAAACAGGTAAAAAAGAC
94N-R:CTTTCGTTTTATTTGAGATCTTCACAAATCTTCAGAAATGAAAC
94C-F:GTAGTGGTGGCGGCGGGAGCGAAGGTGTTGATGCTACTTTGG
107N-R:CTTTCGTTTTATTTGAGATCTTCAATTGTTAGAATGAACAACCAAAG
107C-F:GTAGTGGTGGCGGCGGGAGCTTTACTAATACTATTTTAGAAG
the constructed split-HRV 3C protease recombinant vector and HRV 3C protease substrate expression vector with three split positions of K92, L94 and N107 are co-transformed into an Escherichia coli BL21(DE3) expression host respectively according to the method described in example 1, and after induced expression is carried out according to the method described in example 1, samples are detected by using a CytoFLEX flow cytometer, and the results show that the split-HRV 3C proteases with the three split positions of K82, L94 and N107 can be reassembled to form the HRV 3C protease with complete functional activity, as shown in FIG. 5, wherein K82 is the optimal split position, 85-97% of cells expressing the split-HRV 3C (K82) protease show green fluorescence, and the green fluorescence is close to wild-type HRV 3C protease (98% of green fluorescence cells).
Selecting split-TEV protease as a control, and comparatively testing the working effects of the split-HRV 3C (K82) protease and the split-TEV protease, as shown in FIG. 6-FIG. 7, the split-HRV 3C (K82) protease exerts better working effects than the split-TEV protease in the SPEC system at different temperatures (37,30,25 and 18 ℃) under the condition that the SPEC system also comprises different pairs of strong interaction proteins (Cas 1/Cas2-3 from prokaryote Zymomonas mobilis and Yae1/Lto1 from eukaryote Saccharomyces cerevisiae); it is also noteworthy that the highest green fluorescent protein expression level exhibited by the recombinant split-HRV 3C (K82) protease was more than four times higher than that of the recombinant split-TEV protease at 30,25 and 18 ℃, demonstrating that the split-HRV 3C (K82) protease is highly sensitive over a broad temperature range.
Example 3
Construction process of BL21(DE3) -SPEC recombinant strain
To simplify the manipulation and increase the stability of the SPEC system, the sfGFP-LEVLFQGP-SsrA substrate fragment was integrated into the chromosome of E.coli BL21(DE3) using CRISPR/Cas9 coupled with lambda-red recombination technology. During the integration process, pKD46-cas9-gRNA thermo-sensitive vector is required to be constructed and target fragment LpxM-L-sfGFP-LEVLFQGP-SsrA-LpxM-R is required to be prepared.
The primers were designed as follows:
1. amplification of cas9 and annealing to yield gRNAs
CAS9-F:GATATACCATGGATAAGAAATACTCAATAGGCTTAGATATCGGCAC
CAS9-R:CCATCACCTTCCTCTTCTTCTTGGGGTCACCTCCTAGCTGACTCA
gRNA1:CGTCTGCATGCGAGAAATGA
gRNA3:CAGCATGGCAGGAATATCGA
2. Amplifying LpxM-L-sfGFP, sfGFP-LEVLFQGP-SsrA and SsrA-LpxM-R, fusing and amplifying to obtain complete target fragment LpxM-L-sfGFP-LEVLFQGP-SsrA-LpxM-R
LSR-1F:GGGACGCGCAAGCTTCTCGAGATGGAAACGAAAAAAAATAATAG
LSR-1R:GACTGAGCTAGCCGTAAACCATTTTCTGCCCTTGCGA
Sfgfp-F:TTTACGGCTAGCTCAGTCCTAGGTACAATGCTAGCATGGTGAGCAAGGGCG
Sfgfp-R:
CATCGTTAGCTGCTGGTCCTTGGAATAGGACTTCAAGCTTGTACAGCTCGTCC
LSR-2F:
GGACCAGCAGCTAACGATGAAAACTATGCTTTGGCAGCCCAGCGATGTTCCATAA
LSR-2R:GAAAACTGTCCATACCCATGGCTCGAGTTATTTGATGGGATAAAGATC
As shown in FIG. 8, the amplified target fragment LpxM-L-sfGFP-LEVLFQGP-SsrA-LpxM-R and pKD46-cas9-gRNA co-transform E.coli BL21(DE3) which is spread on LA plate and cultured at 30 ℃, pKD46-cas9, pKD46-cas9-gRNA is selected as a control, when cas9-gRNA exerts editing action, cells containing cas9-gRNA will die because the genome is destroyed, cells containing cas9-gRNA will grow normally, and E.coli cells co-transforming cas9-gRNA and the target fragment will survive and be subjected to colony PCR detection and sequencing analysis to screen correct transformants (inserting sfGFP-LEVLFQGP-SsrA sequence into the lpM site on the genome).
Since the temperature-sensitive vector pKD46-cas9-gRNA was also present in the recombinant strain, the correct BL21(DE3) recombinant strain (lpxM:: sfGFP-LEVLFQGP-SsrA) was subsequently cultured at 42 ℃ for 12-16h for plasmid elimination. The strain which was selected to grow on LB plate but not on LA plate was BL21(DE3) (lpxM:: sfGFP-LEVLFQGP-SsrA) recombinant strain with successful plasmid elimination, and was named BL21(DE3) -SPEC.
Example 4
Evaluation of the working Effect of the final SPEC System Using split-HRV 3C (K82) protease in BL21(DE3) -SPEC recombinant strains at different temperatures (37,30,25 and 18 ℃), respectively
The split-HRV 3C (K82) protease series vectors (SPEC-VD and SPEC-VE) are respectively co-transformed into a BL21(DE3) -SPEC host, induced expression is carried out by adopting the method described in example 1 at different temperatures (37,30,25 and 18 ℃), and the result is detected by a CytoFLEX flow cytometer. As shown in FIG. 9, different pairs of strongly interacting proteins (Cas 1/Cas2-3 from zymomonas mobilis and Yae1/Lto1 from saccharomyces cerevisiae) were tested to exhibit the highest green fluorescent protein expression levels at different temperatures (37,30,25 and 18 ℃) for about 4,6,8 and 16h, respectively, indicating that the split-HRV 3C (K82) protease still exhibits good functional activity and sensitivity in the final SPEC system.
The system is a high-sensitivity method which is simple to operate and can be used for quantitatively detecting and analyzing the protein interaction of prokaryotic or eukaryotic species sources, and has wide temperature range adaptability. In addition, given that the split-HRV 3C (K82) protease has better functional activity and sensitivity than the split-TEV protease, the split-HRV 3C (K82) protease can also replace the split-TEV protease, and is widely applied to other split-protease related technologies. And the ClpXP-SsrA degradation pathway is widely present in other bacterial species (such as Bacillus subtilis and the like), the SPEC system method can also be easily transplanted to other bacterial species for study.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.
Claims (10)
1. The protein interaction detection method based on the split-HRV 3C protease is characterized by comprising the following steps:
step 1, fusing superfolder green fluorescent protein sfGFP at the N end of HRV 3C protease substrate sequence LEVLFQ ↓ GP, and fusing SsrA degradation label at the C end of HRV 3C protease substrate sequence to obtain HRV 3C protease substrate polypeptide sfGFP-LEVLFQGP-SsrA;
step 2, integrating sfGFP-LEVLFQGP-SsrA on a chromosome of the escherichia coli to obtain an escherichia coli recombinant strain;
step 3, splitting the HRV 3C protease into an N-terminal HRV 3C protease domain and a C-terminal HRV 3C protease domain, and fusing the capture protein to the N-terminal HRV 3C protease domain to obtain a first recombinant protein; fusing the bait protein to a C-terminal HRV 3C protease domain to obtain a second recombinant protein;
and 4, transferring the first recombinant protein and the second recombinant protein into an escherichia coli recombinant strain for co-expression, detecting a green fluorescent signal of the recombinant escherichia coli cell by using a flow cytometer, if the green fluorescent signal is detected, indicating that interaction exists between the capture protein and the bait protein, and if no green fluorescent signal is detected, indicating that interaction does not exist between the capture protein and the bait protein.
2. The method for detecting protein interaction based on split-HRV 3C protease as claimed in claim 1, wherein the cleavage site of HRV 3C protease in step 2 is K82 site.
3. The split-HRV 3C protease-based protein interaction assay of claim 1, wherein the SsrA degradation tag is recognized by the E.coli endogenous ClpXP degradation complex, resulting in degradation of the SsrA N-terminally fused protein.
4. The split-HRV 3C protease-based protein interaction assay of claim 1, wherein the HRV 3C protease substrate is fused N-terminal to sfGFP and C-terminal to SsrA degradation tag; the HRV 3C protease substrate polypeptide is LEVLFQGP, and the whole fusion protein is sfGFP-LEVLFQGP-SsrA.
5. The split-HRV 3C protease-based protein interaction assay of claims 1-2, wherein the capture and bait proteins are the target protein pair for which interaction is to be studied; the capture protein is fused at the N-terminal HRV 3C protease domain, and the bait protein is fused at the C-terminal HRV 3C protease domain.
6. The method for detecting protein interaction based on split-HRV 3C protease, according to claim 1, wherein when the capture protein and the bait protein interact, the HRV 3C protease at the N-terminal and the C-terminal are fused to form HRV 3C protease with complete function, and the cleavage recognition of the corresponding substrate polypeptide is performed, so that sfGFP signal accumulation and cell fluorescence intensity increase are caused.
7. The split-HRV 3C protease-based protein interaction assay of claim 1, wherein the sfGFP signal can be detected by flow cytometry.
8. An application of a split-HRV 3C protease-E.coli ClpXP system in detecting whether proteins interact and the interaction strength is disclosed, wherein the split-HRV 3C protease-E.coli ClpXP system combines the functions of a ClpXP-SsrA degradation machine, sfGFP and split-HRV 3C protease.
9. A preparation method of an escherichia coli recombinant strain is characterized by comprising the following steps:
step 1, fusing superfolder green fluorescent protein sfGFP at the N end of HRV 3C protease substrate sequence LEVLFQ ↓ GP, and fusing SsrA degradation label at the C end of HRV 3C protease substrate sequence to obtain HRV 3C protease substrate polypeptide sfGFP-LEVLFQGP-SsrA;
and 2, integrating sfGFP-LEVLFQGP-SsrA on a chromosome of the escherichia coli to obtain an escherichia coli recombinant strain.
10. Use of the recombinant strain of escherichia coli prepared by the method of claim 9 for detecting protein interaction and interaction strength.
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