Improved large-scale preparation of phage T4 endonuclease VII overexpressed in E. coli.
Using PCR, we cloned T4 gene 49, which encodes the endonuclease VII, and the inactive mutant gene 49 amE727 into vector pET-11a. In combination with Escherichia coli host strain BL21 (DE3), this system provided excellent repression of the expression of the highly toxic protein before induction with IPTG. After induction, the proteins were made in high quantities while remaining soluble. Dilution of the crude lysate at 1:10,000 continued to show a highly specific activity in the case of the wild-type enzyme. The protein was purified to homogeneity with a recovery of 33% using two chromatography steps. The yield was 20 times higher and the specific activity 500 times higher than that obtained by using the previously published protocol.
Golz
S
S
Institut für Genetik, Universität zu Köln, Germany.
Birkenbihl
R P
RP
Kemper
B
B
D017122:Bacteriophage T4 / Q000201:enzymology*; D017123:Bacteriophage T7 / Q000235:genetics; D001483:Base Sequence; D003001:Cloning, Molecular; D004247:DNA / Q000378:metabolism; D004706:Endodeoxyribonucleases / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D004790:Enzyme Induction; D004926:Escherichia coli / Q000235:genetics; D005814:Genes, Viral / Q000235:genetics; D005822:Genetic Vectors / Q000235:genetics; D007544:Isopropyl Thiogalactoside / Q000494:pharmacology; D008969:Molecular Sequence Data; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D013379:Substrate Specificity; D015678:Viral Structural Proteins / Q000235:genetics
D011993:Recombinant Fusion Proteins; D015678:Viral Structural Proteins; D007544:Isopropyl Thiogalactoside; D004247:DNA; D004706:Endodeoxyribonucleases; C035571:endodeoxyribonuclease VII