以蛋白質突變技術增強Sortase A之轉肽活性

Abstract

轉肽酵素(Sortase)，原屬於原核生物的表面修飾蛋白，藉由辨認特定氨基酸序列，將特定多肽修飾在自身細胞壁之上，使得自身細胞壁修飾各類多肽而產生感染及致病性。了解此轉肽酵素的辨認特質以及其接合能力可望進一步被應用於材料表面之生物修飾上。為此，本研究將著重於此酵素的催化功能和部分胺基酸殘基對反應活性的影響，進一步尋找能促進酵素催化活性的轉肽突變酵素。藉由轉肽酵素之結構和與受質之結合模擬，我們進行多株多點突變， P94S/D160N/D165A/K196T (SrtPro) SrtPro_I181R、 SrtPro_G191S、 SrtPro_R197G 之建構和純化。由水解和接合活性分別測試突變酵素之活性，其中除SrtPro_R197G外，突變株之催化活性均優於野生株，其水解反應速率可增強1.65-5.71倍；接合反應接合速率則可增強3.71-6.54倍。但相對於野生株，SrtPro_R197G之水解速率為野生株之38%及接合速率為22 %。此實驗證實了四點突變SrtPro有增進其酵素活性，並發現新增G191S突變點 (即SrtPro_G191S)可使接合速率較野生株增加約6.5倍。其中R197則是重要的殘基，一旦改變便會喪失大部分數酵素活性。轉肽酵素對於鈣離子有一定濃度的需求，鈣離子會進而促使反應發生，但Ni2+、Mg2+和Cu2+對促進酵素反應性則不若Ca2+明顯。亦初步利用轉肽酵素協助生物修飾以辨識前降鈣素(procalcitonin, PCT)，並驗證了轉肽酵素後續應用之可行性。

Abstract Sortase refers to a group of prokaryotic enzymes that modify surface proteins by recognizing and cleaving a carboxyl-terminal sorting signal. The enzyme possesses both the peptidase and transpeptidase activity. The recognition signal of sortase consists of the motif LPXTG (Leu-Pro-Any-Thr-Gly). Cleavage occurs between the Thr and Gly, with transient attachment through the Thr residue to the active site of sortase, followed by transpeptidation that attaches a protein covalently to the cell wall. Sortases occur in almost all Gram-positive bacteria. That make their own infection and pathogenicity of various types by cell wall modification. For learning the transpeptidase activity of SrtA, mutants with multiple mutations, such as P94S/D160N/D165A/K196T SrtPro, (SrtPro_I181R), (SrtPro_G191S), (SrtPro_R197G), have been constructed and purified. Except for SrtPro_R197G mutant, the hydrolytic activity and the traspeptidease activity of mutants are 1.65-5.71-fold and 3.71-6.54-fold, respectively, faster than those of the wild-type enzyme. Yet, the mutant SrtPro_R197G shows weaker activity on both hydrolysis and ligation with 38% and 22% of that of the wild-type enzyme. In addition, Ca2+ was confirmed to to be essential for enzymatic activity, whereas Ni2+, Mg2+, and Cu2+ were also shown to have weaker effect to promote the enzymatic activity. The application of sortase on bio-conjugation of a scFv to against procalcitonin is also demonstrated in this study. We thus nconfirm the potential application of sortase for biosening research.