Construction Of Yeast Displaying System Of Novel Anti-infective Target Sortase A

The Sortase A with catalytic function of membrane-bound transpeptidase could link a variety of cell surface pathogenic proteins of gram-positive bacteria which were synthesized in the cytoplasm to cell wall. In recent years, the establishment of an effective high-throughput assay of sortase A could help us to screen sortase A's inhibitors. Because the assay could search a safe and effective solution for pathogen resistance, it became a research focus in biochemistry.Objective:The substrates of sortase A sequence QALPETGEE was introduced to E.coli- Pichia pastoris shuttle expression vector pKFS using EGFP for the detection of its expression. Sortase A activity Construction system of yeast displaying vector pKFS-QALPETGEE-EGFP could easily to detect the Sortase A activity.Methods:(1) According to the sequence of EGFP label gene reported by GenBank, we could design the primers and the EGFP fragment was amplified by PCR using pcDNA/myc-his-EGFP as a template.(2) After being TA-cloned, restriction enzyme digested and sequenced, the QALPETGEE-EGFP was subcloned into E.coli-Pichia pastoris shuttle expression vector pKFS, and named pKFS-QALPETGEE-EGFP.(3) The recombinant strain GS115/ pKFS-QALPETGEE-EGFP was obtained when the plasmid pKFS-QALPETGEE-EGFP was transformed into Pichia pastoris cell and detected expression stability by methanol induced-expression.(4) The prokaryotic expression vector pTRX-srtA was transformed into BL21(DE3)competent cell and induced in diffierent temperature and time to optimize expression product of sortase A.(5) The yeast displaying system of sortase A's substrate was interacted with sortase A to identify the validity of detecting sortase A activity which was based on technology of yeast surface displaying system.Results: (1) A cloning vector pMD19-QALPETGEE-EGFP was obtained.(2) The recombinant GS115/pKFS-QALPETGEE-EGFP was obtained by construction of the expression vector pKFS-QALPETGEE-EGFP and chemical transformation to P. pastoris GS115, which was continuous cultured under methanol induction of 7 days. The results indicated that pKFS-QALPETGEE- EGFP can be used as a stable system for sortase activity assay, as we observed from fluorescence microscope, the intensity of EGFP green fluorescence with time were growing and genetic stablity.(3) The sortase A was obtained under the optimal induced-expression condition of 28℃, 8h induction or 30℃, 8h induction, and interacted with the sortase A's substrate of yeast surface displaying. The results from flow cytometry and fluorescence spectrophotometer indicated that yeast surface displaying system of GS115/pKFS-QALPETGEE-EGFP can be used for Sortase A activity of high-throughput assay.