High level expression of His-tagged colicin 5 in E. coli and characterization of its narrow-spectrum bactericidal activity and pore-forming action.
Since antibiotics with a broad spectrum of activity would select for resistance among the normal flora, colicins having a narrow spectrum of activity can potentially be developed as novel antibiotics. Colicin-based bactericidal proteins with modified spectra of activity might also be developed by further gene fusion or gene modification. To achieve these goals, it is necessary to first build an efficient system to produce large amounts of colicin. In the presence of an immunity gene, we successfully constructed an expression vector pQE30-cfa-cfi producing high levels of His-tagged colicin 5 (60-80 mg/L). We found that the purified His-tagged colicin 5 possesses narrow-spectrum bactericidal activity against nonimmune Escherichia coli cells. It is highly toxic to sensitive E. coli cells at a low concentration of 0.01 microg/ml, while it is nontoxic to other tested gram-negative bacteria, gram-positive bacteria and yeast at a high concentration of 1000 microg/ml. His-tagged colicin 5 kills sensitive cells by permeabilizing their cell membranes. It is not hemolytic to rabbit erythrocytes and has no obvious cytotoxicity to other nucleated mammalian cells at a high concentration of 500 microg/ml. The His-tagged colicin 5 is similar to wild-type colicin 5 in spectrum and bactericidal activity against E. coli. It is a potential novel antibiotic particularly for treating human and animal infections caused by pathogenic E. coli. Besides producing high level of colicin 5, the highly efficient expression vector constructed here might also be a useful tool to develop colicin-based artificial bactericidal proteins.
Yang
Hao
H
Key Laboratory of Transplant Engineering and Immunology, Ministry of Health, West China Hospital, Sichuan University, Chengdu 610041, PR China.
Wan
Lin
L
Li
Xiaowei
X
Cai
Huawei
H
Chen
Lihong
L
Li
Shengfu
S
Li
Youping
Y
Cheng
Jingqiu
J
Lu
Xiaofeng
X
D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics; D002463:Cell Membrane Permeability / Q000187:drug effects; D003001:Cloning, Molecular; D003087:Colicins / Q000096:biosynthesis* / Q000235:genetics / Q000494:pharmacology*; D004926:Escherichia coli / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000096:biosynthesis* / Q000494:pharmacology*; D006461:Hemolysis / Q000187:drug effects; D006639:Histidine / Q000737:chemistry; D008826:Microbial Sensitivity Tests; D011817:Rabbits
D001426:Bacterial Proteins; D003087:Colicins; D029968:Escherichia coli Proteins; C096880:colicin 5 protein, E coli; C007104:colicin immunity proteins; D006639:Histidine