Overproduction and purification of the B2 subunit of ribonucleotide reductase from Escherichia coli.
The nrdB gene of Escherichia coli, coding for the B2 protein of ribonucleotide reductase, has been cloned in a runaway-replication vector. The runaway derivative pBEU17 carries the promoter-proximal portion of the E. coli alanyl-tRNA synthetase gene and proved useful for expressing cloned genes lacking their native transcription initiation signals. The alaS promoter is located approximately 500 base pairs upstream of a single BamHI restriction endonuclease cleavage site utilized in the construction of an expression recombinant plasmid, pBS1, for the nrdB product. After 5-h thermal induction of cells carrying the runaway recombinant pBS1, protein B2 constituted 40% of the soluble protein fraction of the cells. The high concentration of protein B2 in crude extracts of induced cells has enabled a simplified purification scheme to be developed for production of homogeneous and concentrated B2 preparations. Protein B2 produced from pBS1 is identical to the chromosomally encoded nrdB product of E. coli as regards molecular mass on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, enzyme activity, tyrosine radical content, and structure of the binuclear iron center. Amino acid sequence analysis showed that the two polypeptide chains of protein B2 are identical. They start with an alanine residue, and the first 30 residues confirmed the amino acid sequence predicted from the nucleotide sequence of the nrdB gene, apart from an NH2-terminal processing removal of the initiator methionine.
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000096:biosynthesis; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology*; D006358:Hot Temperature; D046911:Macromolecular Substances; D010957:Plasmids; D012264:Ribonucleotide Reductases / Q000096:biosynthesis*; D013997:Time Factors
D001426:Bacterial Proteins; D046911:Macromolecular Substances; D012264:Ribonucleotide Reductases