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B6db references: 97013824

type Journal Article
authors Iurescia, S.; Condo, I.; Angelaccio, S.; Delle Fratte, S.; Bossa, F.
title Site-directed mutagenesis techniques in the study of Escherichia coli serine hydroxymethyltransferase
journal Protein Expr Purif
ui 97013824
year (1996)
volume 7
number 3
pages 323-8.
keywords Alanine/pharmacology
abstract The 3340-bp fragment containing the Escherichia coli glyA gene coding for serine hydroxymethyltransferase was reduced in size by PCR, and the 1600-bp fragment obtained was cloned into the vector pBR322 in both orientations (5'-3', and 3'-5'). This DNA manipulation allowed us to perform site-directed mutagenesis by PCR on the glyA gene. To overcome the problem of the presence of wild-type protein in the various mutant enzyme preparations, the E. coli strain GS245 used to express recombinant serine hydroxymethyltransferase was made recA deficient through generalized transduction mediated by phage P1. The new strain was used for the production of a mutant form of the enzyme, in which the pyridoxal 5'-phosphate binding lysine was substituted by a glutamine. The preparation of this mutant form was completely devoid of wild-type enzyme contamination and measurements of its catalytic activity in the transamination reactions of L- and D-alanine confirmed the suggestion that the active site lysine is not the base that removes the alpha-proton from the substrate.
last changed 2002/11/04 17:41

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