Cloning, Expression And Purification Of DNA Gyrase GyrB From Helicobacter Pylori And GyrA, GyrB From Escherichia Coli

1 ObjectiveBacterial DNA gyrase is the target of a number of antibacterial agents including the aminocoumarins and quinolones.Aim of the research is to establish a model for screening new gyrase inhibitors from Chinese Medicine,and the objective of the current study is to clone and express DNA gyrase from Helicobacter Pylori(HP) and Escherichia coli(E.coli) as fusion protein.The results,on one hand,will contribute to understanding of the antimicrobial mechanism of Chinese Medicine.On the other hand,it may lead to development of novel therapeutic agents.2 Methods2.1Amplification of the genes of interestSpecific PCR primers were designed,and DNA gyrase gyrB gene of HP J99 strain, gyrA and gyrB genes ofE.coli DH5αstrain were amplified by PCR technique,respectively.2.2Construction of cloning plasmidsPCR products were recovered,purified,and then ligated into T vectors.Ligations were transformed into E.coli DH5αcompetent cells.Transformants were selected by resistance of ampicillin and Blue-and-White Screening,and then positive colonies were further screened and indentified by plasmid DNA extraction,restriction enzyme digestion,agarose electrophoresis,and sequencing.2.3Construction of prokaryotic expression plasmids The cloning plasmids harbouring insert fragments of HP J99 gyrB,E.coli DH5αgyrA and gyrB were double-digested respectively with suitable restriction enzymes selected on purpose.The target DNA fragments were separated by agarose electrophoresis and purified using silicon columns,and then were ligated into the prokaryotic expression vector pRSETB,which was double-digested with the same restriction enzymes.Ligations were transformed into E.coli DH5αcompetent cells.Transformants were selected by resistance of ampicillin,and positive colonies were further screened and indentified by plasmid DNA extraction,restriction enzyme digestion,agarose electrophoresis,and sequencing.2.4Expression and purification of fusion proteinsThe prokaryotic expression plasmids harbouring HP J99 gyrB,E.coli DH5αgyrA and gyrB were transformed respectively into the host strain E.coli BL21(DE3) pLysS. Transformants were screened by resistance of ampicillin and chloramphenicol,and positive colonies were further indentified by plasmid DNA extraction,restriction enzyme digestion and agarose electrophoresis.The genes of HP J99 gyrB,E.coli DH5αgyrA and gyrB were expressed as N-terminal His₆-tag fusion proteins by IPTG induction in E.coli BL21(DE3) pLysS host.SDS-PAGE was used to analyze the results of protein expression,and the Ni²⁺ affinity columns were used for purification of fusion proteins.3 Results3.1PCR amplificationAgarose electrophoresis analysis of PCR products showed that the complete sequence of HP J99 gyrB,E.coli DH5αgyrA and gyrB gene were successfully amplified with the length of 2335bp,2678bp and 2488bp,respectively.3.2Construction of cloning plasmidsAgarose electrophoresis analysis and sequence alignment showed that the cloning plasmids pGEM-HPgyrB,pGEM-ECgyrA and pGEM-ECgyrB harbouring HP J99 gyrB,E. coli DH5αgyrA and gyrB gene fragments,respectively,were successfully constructed.A BLAST of the NCBI database with the nucleotide sequence of these three genes exhibited 99.8%(2317/2322),99.7%(2621/2628) and 99.9%(2412/2415) homologous to the sequence of HP J99 strain gyrB,E.coli K12 strain gyrA and gyrB respectively. 3.3 Construction of prokaryotic expression plasmidsAgarose electrophoresis analysis and sequence alignment showed that the prokaryotic expression plasmids pRSETB-HPgyrB,pRSETB-ECgyrA,pRSETB-ECgyrB harbouring HP J99 gyrB,E.coli DH5a gyrA and gyrB gene fragments,respectively,were successfully constructed.The sequence analysis identified three open reading frames encoding three polypeptides of 773,864 and 804 amino acid which showed 99.5%(768/773),99.4% (859/864) and 99.6%(801/804) homologous to the sequence of HP J99 strain GyrB,E.coli K12 strain GyrA and GyrB respectively.The calculated mass of these three His6-tag fusion proteins were 92.0KDa,100.8KDa and 96.3KDa respectively.3.4 Expression and purification of fusion proteinsSDS-PAGE results showed that there was no obvious bands about 92.0KDa from E. coli BL21(DE3) pLysS host containing pRSETB-HPgyrB after IPTG induction,while the purified products of fusion protein under native conditions and denaturing conditions were approximately 75KDa and 92KDa,respectively,the former differed from the expected size of fusion protein(92.0KDa) to a large extent;and that there was no obvious bands about 100.8KDa from E.coli BL21(DE3) pLysS host containing pRSETB-ECgyrA after IPTG induction,while the purified products of fusion protein under native conditions and denaturing conditions were approximately 80KDa and 95KDa respectively,both differed from the expected size of fusion protein(100.8KDa).Transformation of the prokaryotic expression plasmid pRSETB-ECgyrB into the host strain E.coli BL21(DE3) pLysS was failed,so no further overexpression and purification of E.coli gyrB gene was carried out in the current study.4 ConclusionThree genes,namely gyrB from HP J99 strain,gyrA and gyrB from E.coli DH5αstrain,were cloned into T-vector pGEM-T Easy and prokaryotic expression vector pRSETB successfully.However,overexpression of the three genes in E.coli was unsuccessful.The purified fusion proteins were different from the expected target proteins in size.The reasons may lie in the cell toxicity or instability of the target proteins,which resulted in the low-level expression.Therefore,the methods of culture and protein purification should be further optimized,and higher specific tests such as western-blot or enzyme activity examination should be applied to the protein products.In short,further studies should be carried out to obtain the active bactierial GyrA and GyrB subunits.