酵母表面展示分选酶底物用于分选酶活性检测

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

摘要:

摘要：【目的】以EGFP标签检测分选酶底物QALPETGEE在毕赤酵母表面的表达，然后将酵母表面展示的底物与分选酶相互作用以检测分选酶活性。【方法】以pcDNA-myc-his-EGFP为模板，通过PCR技术将QALPETGEE-linker-EGFP基因连接到穿梭载体pKFS上，构建QALPETGEE-linker-EGFP酵母表面展示载体后转化至毕赤酵母（Pichia pastoris）GS115中。重组菌经培养，利用荧光显微镜检测重组酵母的荧光强度，然后通过荧光分光光度计检测分选酶与底物相互作用后产

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.