A system for heterologous expression and isolation of Escherichia coli RNA polymerase and its components.
A set of plasmid vectors for expression of all major Escherichia coli RNA polymerase subunits as fusion proteins with intein- and chitin-binding domains, allowing protein purification in accordance with IMPACT technology, was constructed. It is demonstrated that the fusion subunits alpha, beta or beta' in conjunction with the natural subunits alpha, beta, beta', and sigma can participate in RNA polymerase assembly in vivo, providing affinity-based isolation of the enzyme. Functional activity of the enzyme preparations was demonstrated in the experiments on in vitro transcription and promoter complex formation. With the use of IMPACT technology, sigma(70) subunit can be isolated as an individual protein without admixture of RNA polymerase.
Khodak
Yu A
YA
Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, Moscow, Russia.
Koroleva
O N
ON
Drutsa
V L
VL
D001483:Base Sequence; D001665:Binding Sites; D002686:Chitin; D012321:DNA-Directed RNA Polymerases / Q000302:isolation & purification / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins / Q000302:isolation & purification / Q000378:metabolism*; D005822:Genetic Vectors / Q000235:genetics / Q000378:metabolism; D047668:Inteins; D008969:Molecular Sequence Data
D029968:Escherichia coli Proteins; D002686:Chitin; D012321:DNA-Directed RNA Polymerases