Soluble expression of cloned phage K11 RNA polymerase gene in Escherichia coli at a low temperature.
The gene 1 of the Klebsiella phage K11 encoding the phage RNA polymerase was amplified using the polymerase chain reaction of the Pfu DNA polymerase, cloned and expressed under the control of tac promoter in Escherichia coli. Although the gene was efficiently expressed in E. coli BL21 cells at 37 degrees C, most of the K11 RNA polymerase produced was insoluble, in contrast to soluble expression of the cloned T7 RNA polymerase gene. Coexpression of the bacterial chaperone GroES and GroEL genes together did not help solubilize the K11 RNA polymerase. When the temperature of cell growth was lowered, however, solubility of the K11 RNA polymerase was increased substantially. It was found much more soluble when expressed at 25 degrees C than at 30 and 37 degrees C. Thus, the cloned K11 RNA polymerase gene was expressed in E. coli mostly to the soluble form at 25 degrees C. The protein was purified to homogeneity by chromatography using DEAE-Sephacel and Affigel-blue columns and was found to be active in vitro with the K11 genome or a K11 promoter. The purified K11 RNA polymerase showed highly stringent specificity for the K11 promoter. Low-level cross-reactivity was shown with the SP6 and T7 consensus promoters, while no activity shown with the T3 consensus promoter at all.
Han
K G
KG
Division of Life Science, Pai-Chai University, 439-6 Doma-dong, Seo-gu, Taejon, 302-735, Korea.
Lee
S S
SS
Kang
C
C
D001483:Base Sequence; D003001:Cloning, Molecular; D003080:Cold Temperature; D017931:DNA Primers / Q000235:genetics; D012321:DNA-Directed RNA Polymerases / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification; D004926:Escherichia coli / Q000235:genetics*; D015870:Gene Expression; D005814:Genes, Viral; D017902:Podoviridae / Q000145:classification / Q000201:enzymology* / Q000235:genetics*; D011401:Promoter Regions, Genetic; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D012995:Solubility; D013045:Species Specificity
D017931:DNA Primers; D011994:Recombinant Proteins; D012321:DNA-Directed RNA Polymerases