Expression,Purification And Characterization Of GST-STAR Fusion Protein In Escherichia Coli

The sortase A(SrtA) of Staphylococcus aureus is a membrane-bound transpeptidase toanchor cell surface proteins which were synthesized in the cytoplasm to the peptidoglycan ofcell wall. The loss or mutation of SrtA can significantly reduce the infection ability ofpathogen, so SrtA can be used as an anti-infection target enzyme for new antibacterial drugdevelopment. In addition, because of SrtA’s specificity and catalytic mechanism, SrtA hasbeen widely used in the field of biotechnology.The aim of the study is to construct the prokaryotic expression vector pGEX-SrtA△N59toexpress the fusion protein GST-SrtA△N59in Escherichia coli, and purify the protein by affinitychromatography.According to the sequence of SrtA gene(AF162687) reported by GenBank, we designedthe primers and amplified the truncated SrtA gene, SrtA△N59, from recombinant vectorpMD20-SrtA by PCR and obtained about460bp DNA fragment. The fragment was cloned tothe expression plasmid pGEX-4T-1. The recombinant plasmid pGEX-SrtA△N59was identifiedby restriction enzyme and sequencing, and the results showed that we had successfullyconstructed expression vector pGEX-SrtA△N59.The recombinant plasmid pGEX-SrtA△N59was transformed into Escherichia coliBL21(DE3) and was induced by IPTG. The expression products were analyzed by SDS-PAGE.The results showed that approximately42KDa GST-SrtA△N59fusion protein was expressed bypGEX-SrtA△N59and the protein was soluble. The expression yield of target protein wasincreased by optimizing induction temperature and time. We purified the fusion protein byglutathione agarose affinity chromatography, the purity was up to98%.The recombinant Pichia pastoris yeast GS115/pKFS-QALPETGEE-EGFP which wasconstructed by our lab could display SrtA’s substate sequence QALPETGEE with EGFP onthe yeast surface. The activity of SrtA could be detected by fluorescence intensity variety offree EGFP in the reaction supernatant. The purified GST-SrtA△N59fusion protein wasinteracted with the substrate displayed on yeast surface, the fluorescence intensity of freeEGFP increased by352.77, this indicated that the fusion protein had high activity. In addition,we compared the activity of recombinant SrtA expressed by pGEX-SrtA△N59and pET32a- SrtA△N59respectively, the results showed that the activity of recombinant SrtA expressed bypGEX-SrtA△N59was much higher.In this study, we successfully constructed the expression vector pGEX-SrtA△N59andobtained high purity, high active GST-SrtA△N59fusion protein by affinity chromatography.This would lay a good foundation for the study of SrtA enzyme properties as well as itsapplication.