High-level expression of a soluble functional antimicrobial peptide, human beta-defensin 2, in Escherichia coli.
In this work, taking human beta-defensin-2 (HBD2) as a demonstrative molecule, the strategies for high efficient production of functional human beta-defensins in E. coli were studied. Fusion mature HBD2 (TrxA-mHBD2) showed high solubility and productivity without the need for lowering the cultivation temperature. The solubility of target fusion protein could attain 81.3% even at 37 degrees C with a volumetric productivity as high as 235 mg/L in a rich medium MBL at the same temperature and reached 346 mg/L at 28 degrees C. The His-Tag in the fusion protein enabled the application of affinity chromatography separation to obtain high purity of the overexpressed recombinant fusion protein. After digestion by enterokinase, purification via cationic exchange chromatography, and desalting by ultrafiltration, mature HBD2 product was obtained with a purity of 95% in an overall recovery of 29.2%. The antimicrobial activity of the recombinant mature HBD2 and the influence factors were tested using E. coli K12D31 as a sensitive strain.
Xu
Zhinan
Z
Institute of Bioengineering, Department of Chemical and Biochemical Engineering, Zhejiang University, Hangzhou 310027, China. znxu@zju.edu.cn
Peng
Li
L
Zhong
Zhixia
Z
Fang
Xiangming
X
Cen
Peilin
P
D000595:Amino Acid Sequence; D000900:Anti-Bacterial Agents / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism* / Q000494:pharmacology; D002470:Cell Survival / Q000187:drug effects; D004926:Escherichia coli / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D015964:Gene Expression Regulation, Bacterial; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism / Q000494:pharmacology; D012995:Solubility; D023083:beta-Defensins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism* / Q000494:pharmacology
D000900:Anti-Bacterial Agents; C106641:DEFB4A protein, human; D011994:Recombinant Proteins; D023083:beta-Defensins