Cloning, expression, and purification of gene 3 endonuclease from bacteriophage T7.
The structural gene for the single-stranded endonuclease coded for by gene 3 of bacteriophage T7 has been cloned in pGW7, a derivative of the plasmid pBR322, which contains the lambda PL promoter and the gene for the temperature-sensitive lambda repressor, cI857. The complete gene 3 DNA sequence has been placed downstream of the PL promoter, and the endonuclease is overproduced by temperature induction at mid-log phase of Escherichia coli carrying the recombinant plasmid pTP2. Despite the fact that cell growth rapidly declines due to toxic effects of the excess endonuclease, significant amounts of the enzyme can be isolated in nearly homogeneous form from the induced cells. An assay of nuclease activity has been devised using gel electrophoresis of the product DNA fragments from DNA substrates. These assays show the enzyme to have an absolute requirement for Mg(II) (10 mM), a broad pH optimum near pH 7, but significant activity from pH 3 to pH 9, and a 10-100-fold preference for single-stranded DNA (ssDNA). The enzyme is readily inactivated by ethylenediaminetetraacetic acid or high salt. The differential activity in favor of ssDNA can be exploited to map small single-stranded regions in double-stranded DNAs as shown by cleavage of the melted region of an open complex of T7 RNA polymerase and its promoter.
Pham
T T
TT
Coleman
J E
JE
D010582:Bacteriophage lambda / Q000235:genetics; D003001:Cloning, Molecular; D004722:Endoribonucleases / Q000235:genetics*; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D005796:Genes; D005814:Genes, Viral; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D008715:Methionine / Q000378:metabolism; D010957:Plasmids; D014176:Protein Biosynthesis; D012965:Sodium Chloride / Q000494:pharmacology; D013604:T-Phages / Q000201:enzymology / Q000235:genetics*; D014158:Transcription, Genetic
D012965:Sodium Chloride; D008715:Methionine; D004722:Endoribonucleases; C036544:endoribonuclease, single-stranded specific; D008274:Magnesium