Expression and bioactive characterization of bacteriophage lysin gene of Bacillus anthracis in Escherichia coli / 生物工程学报

ABSTRACT

The lysin gene of Bacillus anthracis-diagnosing bacteriophage, obtained by PCR amplification,was cloned into the Escherichia coli exepression vector pET22b which has been cleaved by EcoR I and Nde I. The recombinant vector pET22b-gamma lysin was verified to be correctly constructed by PCR, sequencing and enzyme digestion, and highly expressed in E. coli BL21 (DE3), which accounted for about 40 percent of total protein in E. coli BL21 (DE3), while in the 5L fermentor the expression level reached 15g/L. After expression, disruption and purification with three-step chromatography, Streamline SP, SP HP and Sephacryl S-100, the recombinant gamma lysin was finally obtained with purity of higher than 95 percent as determined by gel scan. The final yield following SP HP was 19.1 percent, with a greater-than-350-fold increase in specific activity. The pure enzyme has been shown active to Bacillus anthracis, and not to E. coli, Bacillus subtilis and Bacillus cereus. Its specific activity was about 1400 u/mg.