| Summary: | 碩士 === 國立臺灣大學 === 微生物與生化學研究所 === 92 === We obtained Aeromonas hydrophila CKH29 from the patients in National Cheng Kung University. In the previous study, we have cloned and sequenced a proteolytic gene prtS1 of the serine protease, which was highly homologous to HtrA (DegP)/DegQ/DegS family serine protease. Both of the two belonged to trypsin-like protease. In E.coli, HtrA (high-temperature-requir
-ement protein A) is the first known to relate with existence under high temparature (Lipinska, 1990). Moreover, it was observed in the study of S. typhimurium htrA mutant that htrA might also correlate with pathogenicity (Johnson, 1991).
The current study aimed to explore prtS1 and the role and function of its products in two respects. By conjugation and electroporation, we transformed pSup2021-PrtSM into A. hydrophila to perform homologous recombination, so as to find out the influence of prtS1 absence. According to the result, seven serine protease mutants were obtained; while a later PCR indicated that they were not response to prtS. Therefore, we selected KpnI、SalI、PvuI、PstI、PvuII to react upon CKH29 chromosome, and then performed Southern blot to make sure that prtS had only one copy in CKH29.
The behavior of His-tag PrtS1 fusion protein pPDZ03 in JM109 was used to find out whether there is any protein related to PrtS1 and the role it plays. According to the result, the interference of nonspecific proteins was irresistible by the one-step purification being applied. So further improvement and fusion of the proteins in the host cell is needed in order to explore the related interactive proteins.
In the discussion of the protease active region of PrtS1, it was found that the PDZ domain had something to do with the regulator of protease activity. That is, the lack of two domains would cause PrtS1 to fail to regulate, and then the host was inclined to death. When PDZ domain II was missing, the protease would lose the activity; when PDZ domain I was missing, the activity would decrease. We conclude that PDZ domain II might correlate to the recognition of specific substrates and that PDZ domain I might correlate to the formation of appropriate polymer. The center of serine protease related to PrtS1 activity still had protease activity after site mutating at the 214th serine protease. Besides, a similar sequence was observed to occur at the 211th serine protease, and it might be the center of another PrtS1.
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