Construction, expression, and preliminary characterization of chimeric class μ glutathione S-transferases with altered catalytic properties

✍ Scribed by Pinghui Zhang; Richard N. Armstrong

• Book ID: 102763562
• Publisher: Wiley (John Wiley & Sons)
• Year: 1990
• Tongue: English
• Weight: 983 KB
• Volume: 29
• Category: Article
• ISSN: 0006-3525
• DOI: 10.1002/bip.360290121

✦ Synopsis

Abstract

An expression plasmid for isoenzyme 3–3 of rat liver glutathione S‐transferase has been constructed from the cDNA clone pGTA/C44 and the pAS expression vector pMG27NS, and used for the efficient production of the enzyme in the Escherichia coli strain M5219. The plasmid has also been manipulated, through theuse of synthetic linkers, to encode chimeric polypeptides in which short sequnces of the closely related isoenzyme 4–4 have been substituted into the N‐terminal and C‐terminal variable domains of isoenzyme 3–3. The chimeric polypeptides designated 4^9^3^208^, 3^209^4^8^, and 4^9^3^200^4^8^ are expressed with varying degrees of efficiency in E. coli. The active dimeric holoenzymes 3‐3, (4^9^3^208^)~2~, (3^209^4^8^)~2~, and (4^9^3^200^4^8^)~2~ can be isolated. The spectroscopic and kinetic properties of the chimeric enzymes are significantly different than the native enzyme.