Recombinant RNA polymerase: inducible overexpression, purification and assembly of Escherichia coli rpo gene products.
The genes, rpoA, rpoB and rpoC of Escherichia coli, which encode the RNA polymerase alpha-, beta- and beta'-subunits, respectively, have been individually placed on expression plasmids under the control of the bacteriophage T7 promoter. Induction of the T7 RNA polymerase gene in host cells harboring each of the three plasmids resulted in the extensive overproduction of the three polypeptides. The overproduced subunits were purified and assembled into a functional enzyme, whose specific activity and dependence on the sigma-factor were indistinguishable from native RNA polymerase purified by conventional methods.
Zalenskaya
K
K
Department of Microbiology, Columbia University College of Physicians and Surgeons, New York, NY 10032.
Lee
J
J
Gujuluva
C N
CN
Shin
Y K
YK
Slutsky
M
M
Goldfarb
A
A
D003001:Cloning, Molecular / Q000379:methods; D012321:DNA-Directed RNA Polymerases / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D015870:Gene Expression; D005798:Genes, Bacterial; D007700:Kinetics; D046911:Macromolecular Substances; D010957:Plasmids; D011401:Promoter Regions, Genetic; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification; D013604:T-Phages / Q000235:genetics
D046911:Macromolecular Substances; D011994:Recombinant Proteins; D012321:DNA-Directed RNA Polymerases