Abstract
The Escherichia coli biotin operon repressor protein (BirA) has been overexpressed at the level of 0.5-1% of the total cellular protein from the plasmid pMBR10. Four lines of evidence demonstrated that authentic BirA protein was produced. First, birA plasmids complemented birA mutants for both the repressor and biotin holoenzyme synthetase activities of BirA. Second, biotin holoenzyme synthase activity was increased in strains containing the overproducing plasmids. Third, deletion of sequences flanking the birA gene did not alter production of the 35-kDa BirA protein, but insertion of oligonucleotide linkers within the birA coding region abolished it. Fourth, the 35-kDa protein copurified with the biotin binding activity normally associated with BirA. The birA protein has been purified to homogeneity in a three-step process involving chromatography on phosphocellulose and hydroxyapatite columns.
Highlights
The Escherichia colibiotin operon repressor protein boxylase]synthetase (EC 6.3.4.15)
The36-kDa protein copurified with the biotin binding activity normally associatedwith BirA
The biotinyl-5’-adenylate acts as the corepressor for repression of the biotin operon [2,13].Because biotin transfer is the final step in the utilization of biotin as an enzyme cofactor, it is a logical control point for the
Summary
Gene, designated birA or bioR [1,2, 6, 7,8,9]. Genetic (1,2, lo), biochemical [3], andnucleotide sequence studies [11,12]have.
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