Open AccessCloning, overproduction, and purification of the B2 subunit of ribonucleoside-diphosphate reductase
Author(s) -
Scott P. Salowe,
JoAnne Stubbe
Publication year - 1986
Publication title -
journal of bacteriology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.652
H-Index - 246
eISSN - 1067-8832
pISSN - 0021-9193
DOI - 10.1128/jb.165.2.363-366.1986
Subject(s) - biology , thermolabile , overproduction , ribonucleotide reductase , escherichia coli , protein subunit , microbiology and biotechnology , plasmid , lysogenic cycle , structural gene , pbr322 , repressor , bacteriophage , gene , biochemistry , molecular cloning , complementary dna , enzyme , gene expression
The nrdB gene, which encodes the B2 subunit of Escherichia coli ribonucleotide reductase (EC 1.17.4.1), was cloned into multicopy plasmid pSPS2. This vector, which contains the pL promoter of bacteriophage lambda and the tetracycline resistance gene of pBR322, was transformed into a lysogenic host with a thermolabile repressor. In the newly constructed strain, subunit B2 constituted approximately 25% of the soluble protein after heat induction, an overproduction of several hundredfold relative to the wild-type strain. Purification to homogeneity of the overproduced protein was accomplished by using DEAE and quaternary aminoethyl ion-exchange resins.