Site-directed mutagenesis of rat thioltransferase: Effects of essential cysteine residues for the protection against oxidative stress
✍ Scribed by Tomoyuki Terada; Kei-ichi Okamoto; Jun-ichi Nishikawa; Takeshi Miura; Toru Nishinaka; Tsutomu Nishihara
- Publisher
- John Wiley and Sons
- Year
- 2010
- Tongue
- English
- Weight
- 116 KB
- Volume
- 24
- Category
- Article
- ISSN
- 1095-6670
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✦ Synopsis
Abstract
A cDNA of rat liver thioltransferase was cloned and then expressed using pMAL‐c expression vector in Escherichia coli. Recombinant rat liver thioltransferase was expressed as a fusion protein with maltose‐binding protein and then purified by amylose resin column chromatography to be homogeneity on 12.5% SDS‐polyacrylamide gel electrophoretic analysis. The expressed proteins were shown as two bands at around 53 and 41 kDa, suggesting that the high molecular one was a fusion protein of recombinant thioltransferase (11.7 plus 41 kDa) and the other (smaller one) was a maltose‐binding protein (41 kDa). A recombinant thioltransferase catalyzed a thiol/disulfide exchange reaction in the same way as thioltransferases purified from various sources. Compared with wild type, the mutants C23A, C26A, C79A, and C83A showed 0%, 17%, 82%, and 86% in the enzymatic activity, respectively. In addition, wild‐type‐transfected bacteria expressed in bacterial cells showed a strong resistance to H~2~O~2~ treatment as well as the case of active mutants (C79A and C83A), but inactive mutants (C23A and C26A) showed no resistance to H~2~O~2~ treatment as same as mocktransfection. Thioltransferase can be important for survival of bacterial cells under oxidative stress. © 2010 Wiley Periodicals, Inc. J Biochem Mol Toxicol 24:60–65, 2010; Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/jbt.20312