Studies on the sortase activity assay via display its substrates on yeast surface

Abstract:

Abstract：[Objective] QALPETGEE sortase’s substrate was anchored on the cell surface of yeast Pichia pastoris using EGFP for the detection of its expression. The sortase activity assay is based on interaction of sortase and substrate displaying on yeast. [Methods] The gene-encoding QALPETGEE- linker-EGFP was amplified by PCR using pcDNA/myc-his-EGFP as a template, and then inserted into shuttle vector pKFS. Next, the vectors were introduced into Pichia pastoris GS115. After cultivation, recombinant cells was verified with fluorescence microscopy and sortase activity was detected by fluorescence spectrophotometer from variety of free EGFP’s flurescence intensity. [Results] The green cells were observed by fluorescence microscopy, enhancing over time. Fluorescence spectrophotometer convinced that fluorescence intensity of free EGFP in the reaction supernatant has increased from 187.67±2.16 to 273.47±2.14 after interaction of sortase and its substrates. [Conclusion] The result suggests that sortase’s substrates have been display on yeast successfully, which could use for sortase activity assay high-effectively and economically.

Key words:Keywords: yeast display, sortase; fluorescence spectrophotometer; substrate; activity assay