探討Sortase A突變株D165A、K196T和P94S/D160N/D165A/K196T的反應性

Abstract

轉肽酶Sortase A藉由特殊的胺基酸序列－N端的LPETG序列與C端的GGGGG序列，可將兩端不同功能的蛋白質或胜肽段連接起來，同時保持正確的構形，形成多功能嵌合蛋白。本研究藉由兩種不同的螢光受質Abz-LPETG-Dnp與GGGGGK-FITC，來描述Sortase A野生株與突變株的水解及接合之功能。發現Sortase A的P94S、D160N、D165A、K196T四點突變株比Sortase A的野生株在水解初速率上大3.14倍，在接合初速率上大1.18倍。同時應用Sortase A的作用，將GGGGGK-FITC的胜肽段接合在具有LPETG的QCM晶片載體上。未來可擴大此應用在具有LPETG的晶片載體上，接合不同的抗體作探針進行免疫檢查。

Sortase A is kind of transpeptidase which can connect two different function proteins or peptides together to form the multifunction chimeric protein without interrupt the structure and function by using the special amino acid sequences: the N terminal LPETG and the C terminal GGGGG. In this study, we compare the function of hydrolysis and connection between the wildtype and mutant line by using two different fluorescence substrates the Abz-LPETG-Dnp and GGGGGK-FITC. We discover the P94S, D160A, D165A, K196T 4 point mutant which is 3.14 times faster in hydrolysis and 1.18 times faster in connection initial rate than the wildtype sortase A. By sortase A, we can link the protein or peptide with GGGGGK-FITC sequence to the QCM chip with LPETG sequence; using this method, we can link the antibodies to a chip to do the immunoassays.