碩士 / 國立海洋大學 / 水產生物技術研究所 / 89 / Superoxide dismutases (SOD) are metalloenzymes and can detoxify superoxide radicals present in various living cells. VmiMn-SODsfc0 gene was amplified from Vibrio mimicus chromosome DNA by PCR using the primers desinged from GenBank database. There are some differences in nucleotide sequence between VmiMn-SODsfc0 and VmiMn-SODkcy. The shuffled five genes of VmiMn-SODsfc2, 3, 4, 5, 6 were obtained from these two Mn-SODs genes. There are 9 ~ 11 point mutations in four shuffled Mn-SOD gnens and 2 point mutations in one shuffled Mn-SOD gene. The shuffled genes were over-expressed in the heterologous host E.coli BL21(DE3)pLysS using the pET20b(+) expression vector. All the shuffled Mn-SOD proteins are enzymatically active.
The deletion 1 and deletion 2 mutants of Mn-SOD based on the 3’-end of the VmiMn-SODsfc0 gene were made after amino acid sequence homology analysis from the GenBank Mn-SODs from different species. The deletion mutants were heterologously expressed. The deletion 1 mutant Mn-SOD proteins are active, however deletion 2 mutants are all inactive and are present in inclusion bodies.
The engineered recombinant Mn-SOD proteins were efficiently purified by His-tag affinity chromatography. All recombinant proteins were thermo-stable between 45℃ ~ 55℃ and were insensitive to inhibitors such as sodium azide (NaN3) and diethyldithiocarbamic acid (DDC) with concentrations up to 20 mM and 15 mM, respectively.
| Identifer | oai:union.ndltd.org:TW/089NTOU0613002 |
| Date | January 2001 |
| Creators | Fan Chang Shue, 許芳誠 |
| Contributors | Lin Fu Pang, 林富邦 |
| Source Sets | National Digital Library of Theses and Dissertations in Taiwan |
| Language | zh-TW |
| Detected Language | English |
| Type | 學位論文 ; thesis |
| Format | 100 |
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