嗜水性產氣單胞桿菌絲胺酸蛋白酶PrtS1結合蛋白之探討

Abstract

本研究中先前由成功大學取得臨床分離株 Aeromonas hydrophila CKH29，由其中選殖出絲胺酸蛋白酶基因 prtS1。由 prtS1基因之序列分析可知，PrtS1與 E. coli之 HtrA (DegP)/DegQ/DegS family 之 serine protease 有極高的類似性，應屬於 trypsin-like protease。E. coli之 HtrA (high-temperature-requirement protein A) 已報告與菌株在高溫下之生存有關。為明瞭 PrtS1 在A. hydrophila菌株中之功能，本研究中探討其結合相關之蛋白質。研究中以其活性中心突變之PrtS1(S211•214A) 接於6 × His tag 片段成融合蛋白質，以E. coli JM109表現後，使融合蛋白質吸附於Ni-NTA agarose gel上，再以之與 A. hydrophila CKH29 株細胞內容物作用，使融合蛋白與細胞質蛋白作用，尋找可能與之結合的相關蛋白質。蛋白質作用後以含urea及高濃度 imidazol 溶液流洗，使結合之蛋白質分離，再以SDS-PAGE 分析結合之蛋白質。結果可分離到 84 kDa 與 57 kDa 大小之蛋白色帶。為確定蛋白色帶之蛋白質為PrtS1 相關之結合蛋白，再以 LC/MS/MS 分析蛋白之胜肽序列，由序列比對 Aeromonas hydrophila ATCC 7966 genomic sequences 之 Data base，結果發現 A. hydrophila ATCC 7966 之中亦存在相同序列，因此，以該 Data base 之序列設計PCR 之引子，選殖 A. hydrophila CKH29 株中 PrtS1 結合相關蛋白之基因，再經 yeast two-hydrid analysis確定選殖基因之蛋白質與PrtS1之結合。在上述實驗分析中，顯示A. hydrophila CKH29 株中之glycogen branching enzyme、aldehyde dehydrogenase、及 pyruvate kinase為PrtS1可能之結合相關蛋白。由結合相關蛋白之功能推測，PrtS1 可能為菌株處於無氧環境下，維持醣類代謝穩定相關的蛋白酶。

In the previous studies, a serine protease gene prtS1 has been cloned from Aeromonas. hydrophila CKH29, which is a clinical isolate obtained from National Cheng Kung University Hospital. PrtS1 is analysed as a trypsin-like protease as it shows high homology to E. coli HtrA (DegP)/DegQ/DegS family protease. HtrA is known as a protein respond to survival of E.coli under higher temparature. Accordingly, in this reaearch we separated and analyzed interaction proteins of PrtS1 to declear its biological function. PrtS1(S211•214A)-His-tagged protein is used as the acting protein, which composed of a 6 × His tag fragment fused with PrtS1(S211•214A), which a mutanted protein in which the active residues, S211 and S214,has been replaced with 211A and 214A. The acting protein was attached on Ni-NTA agarose gel to prepare an affinity column, then incubated with CKH29 cell lysates. The column was eluted with urea buffer as well as high concentration of imidazole to release possible binding proteins within the column. Two bands of protein of 84 kDa and 57 kDa in the SDS-PAGE were obtained from eluent of the column. Aftert analyzing the partial amino acid sequenes by LC/MS/MS, we identified four candidate genes from CKH29, according to the comparison of higher coverage to genomic Data base of A. hydrophila ATCC 7966. Expression plasmids containing each one of the indetified genes or a malS gene of CKH29, which is known as the substracte of HtrA in ATCC7966, was constructed and yeast two-hybrid analysis was performed by using plasmid containing PrtS1(S211•214A) as a bait. The results of yeast two-hybrid assay revealed that glycogen branching enzyme, aldehyde dehydrogenase, and pyruvate kinase may be the PrtS1-interacting proteins, while not the phosphate acetyltransferase and MalS. We propse that PrtS1 may involved in stabilizing carbohydrate metabolism and controlling bacterial growth or responsing to anaerobic environment.