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Overexpression and mutagenesis of the lipoamide dehydrogenase of Escherichia coli



Overexpression and mutagenesis of the lipoamide dehydrogenase of Escherichia coli



Biochemical Journal 256(3): 741-749



A 'split-gene' technique for the overexpression and mutagenesis of the gene encoding the lipoamide dehydrogenase of Escherichia coli was developed in order to overcome the instability problems encountered when attempting to mutate the intact gene. The lipoamide dehydrogenase gene, lpd, was dissected into two fragments which were separately subcloned into M13 vectors for mutagenesis in vitro folllowed by reconstitution in the pJLA504 expression vector under the transcriptional control of the .lambda.PR and .lambda.PL promotes and a temperature-sensitive .lambda. repressor. After thermoinduction, E. coli cells transformed with the plasmid carrying the reconstituted lpd gene contained 4-5 times more lipoamide dehydrogenase activity than is normally found in the wild-type organism. The strategy was used to engineer a Glu-188 .fwdarw. Asp replacement in lipoamide dehydrogenase, and this generated an enzyme with markedly different kinetic properties.

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Accession: 007625592

Download citation: RISBibTeXText

PMID: 3066354

DOI: 10.1042/bj2560741


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