Analysis of interaction protein of Aeromonas hydrophila serine protease PrtS1

• Main Authors: Tang-Yi Huang, 黃堂益
• Other Authors: 劉俊民
• Format: Others
• Language: zh-TW
• Published: 2009
• Online Access: http://ndltd.ncl.edu.tw/handle/92083123067031301812

碩士 === 國立臺灣大學 === 微生物與生化學研究所 === 97 === 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.