Bacteriophage T7 DNA polymerase: cloning and high-level expression.
Phage T7 DNA polymerase consists of a 1:1 complex of the viral T7 gene 5 protein and the host cell thioredoxin. A 3.25-kilobase T7 DNA fragment containing the complete coding sequence of gene 5, and the nearby genes 4.7 and 5.3, was cloned in the BamHI site of the plasmid pBR322. Transformation of the thioredoxin-negative (trxA-) Escherichia coli strain BH215 with the recombinant plasmid pRS101 resulted in large overproduction of gene 5 protein corresponding to a level about 60-fold higher than in T7-infected cells. Transcription of gene 5 probably originates from a previously unknown E. coli RNA polymerase promoter located immediately upstream of the structural gene. Contrary to expectation, pRS101 could be maintained also in E. coli trxA+ cells despite the in vivo formation of active T7 DNA polymerase. However, the expression of gene 5 was lower by a factor of 5-10 than in trxA- cells. Since the plasmid copy number in the two strains was the same, a gene dosage effect can be excluded. The observed difference suggests an autoregulatory interaction of T7 DNA polymerase holoenzyme on the expression of T7 gene 5. The trxA- strain BH215/pRS101 is an excellent source of gene 5 protein and T7 DNA polymerase. After in vitro reconstitution of holoenzyme by addition of excess thioredoxin, highly active T7 DNA polymerase was purified to homogeneity by a simple antithioredoxin immunoadsorbent chromatography technique.
D001483:Base Sequence; D003001:Cloning, Molecular; D004262:DNA Restriction Enzymes; D004259:DNA-Directed DNA Polymerase / Q000235:genetics*; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D005796:Genes; D005814:Genes, Viral; D009693:Nucleic Acid Hybridization; D010957:Plasmids; D013604:T-Phages / Q000201:enzymology* / Q000235:genetics; D014158:Transcription, Genetic
D004259:DNA-Directed DNA Polymerase; D004262:DNA Restriction Enzymes