Improved Large-Scale Preparation of Phage T4 Endonuclease VII Overexpressed in E. coli

Using PCR, we cloned T4 gene 49, which encodes the endonucleaseVII, 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 expressionof the highly toxic protein before induction with IPTG. Afterinduction, the proteins were made in high quantities while remainingsoluble. Dilution of the crude lysate at 1 : 10,000 continuedto show a highly specific activity in the case of the wild-typeenzyme. The protein was purified to homogeneity with a recoveryof 33% using two chromatography steps. The yield was 20 timeshigher and the specific activity 500 times higher than thatobtained by using the previously published protocol.