High-level expression and novel antifungal activity of mouse beta defensin-1 mature peptide in Escherichia coli.
Mouse beta defensin-1 (mBD-1) is a cationic 37-amino acid antimicrobial peptide with three conserved cysterine disulfied bonds. It exhibits a broad antimicrobial spectrum, but mBD-1 against Candida albicans (C. albicans) and Cryptococcus neoformans (C. neoformans) is poorly understood. This study describes the mBD-1 gene, the heterologous fusion expression of the peptide in Escherichia coli, and the bioactive assay of released mature mBD-1. By constructing the expression plasmid (pET32a-mBD1), high yields of soluble mBD-1 fusion protein (0.67 g/L) could be obtained in E. coli and cleaved by enterokinase. The digested product was further purified and desalted with the final amount of pure mature mBD-1 being 0.14 g/L. Classical fungi growth inhibition assay showed clear antifungal activity against C. albicans and C. neoformans with IC(50) of 5 and 2 microM, respectively. The results show that the mBD-1 control fungal colonization through hyphal induction, direct fungicidal activity, and the activity is suppressed by increasing NaCl concentration. Successful expression of the mBD-1 peptide in E. coli offers a basis for further studying its antifungal mechanisms and may provide significance in developing this peptide to an antifungal drug.
Wang
Yueling
Y
Department of Microbiology, West China School of Preclinical and Forensic Medicine, Sichuan University, Chengdu, China.
Jiang
Yan
Y
Gong
Tianxiang
T
Cui
Xiaoyan
X
Li
Wanyi
W
Feng
Yan
Y
Wang
Baoning
B
Jiang
Zhonghua
Z
Li
Mingyuan
M
D000818:Animals; D000935:Antifungal Agents / Q000302:isolation & purification / Q000378:metabolism* / Q000494:pharmacology*; D002176:Candida albicans / Q000187:drug effects; D003455:Cryptococcus neoformans / Q000187:drug effects; D004926:Escherichia coli / Q000235:genetics*; D005434:Flow Cytometry; D015870:Gene Expression; D005786:Gene Expression Regulation; D005818:Genetic Engineering / Q000379:methods*; D051379:Mice; D009928:Organ Specificity; D023083:beta-Defensins / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification / Q000494:pharmacology*
D000935:Antifungal Agents; C120555:Defb1 protein, mouse; D023083:beta-Defensins