Lixin Luo1, Lin Wu, Ying Lin. 1. College of Veterinary Medicine, National Reference Laboratory of Veterinary Drug Residues, Guangdong Provincial Key Laboratory of Veterinary Pharmaceutics Development and Safety Evaluation, South China Agricultural University, Guangzhou 510642, China. btlxluo@scut.edu.cn
Abstract
OBJECTIVE: QALPETGEE sortase's substrate was anchored on the cell surface of yeast Pichia pastoris using EGFP to detect its expression. METHODS: The gene-encoding QALPETGEE-linker-EGFP was amplified by PCR by using pcDNA/myc-his-EGFP as a template, and then inserted into shuttle vector pKFS. Then, the vectors were introduced into Pichia pastoris GS115. After cultivation, recombinant cells were verified with fluorescence microscopy and sortase activity was detected by fluorescence spectrophotometer from variety of free EGFP's fluorescence 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 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 displayed on yeast successfully, which could be used for sortase activity assay.
OBJECTIVE: QALPETGEE sortase's substrate was anchored on the cell surface of yeastPichia pastoris using EGFP to detect its expression. METHODS: The gene-encoding QALPETGEE-linker-EGFP was amplified by PCR by using pcDNA/myc-his-EGFP as a template, and then inserted into shuttle vector pKFS. Then, the vectors were introduced into Pichia pastoris GS115. After cultivation, recombinant cells were verified with fluorescence microscopy and sortase activity was detected by fluorescence spectrophotometer from variety of free EGFP's fluorescence 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 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 displayed on yeast successfully, which could be used for sortase activity assay.