[Expression and bioactive characterization of bacteriophage lysin gene of Bacillus anthracis in Escherichia coli].
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.
Li
Xiao-Jing
XJ
Institute of Biotechnology, Academy of Military Medical Sciences, Beijing 100071, China.
Zhang
Hao
H
Fu
Xue-Qi
XQ
Li
Yan-Ying
YY
Chen
Jing
J
Li
Yu-Ling
YL
Fang
Hong-Qing
HQ
Chen
Hui-Peng
HP
D001408:Bacillus anthracis / Q000821:virology*; D001435:Bacteriophages / Q000201:enzymology*; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D014764:Viral Proteins / Q000235:genetics*
D011993:Recombinant Fusion Proteins; D014764:Viral Proteins