title: Production of bioactive human beta-defensin 5 and 6 in Escherichia coli by soluble fusion expression.
abstract: This work reports the first successful recombinant expression and purification of human beta-defensin 5 (HBD5) and human beta-defensin 6 (HBD6) in Escherichia coli. HBD5 and HBD6 are cationic antimicrobial peptides with three conserved cysteine disulfide bonds. Two codon-optimized sequences coding the HBD5 gene (sHBD5) and HBD6 gene (sHBD6), respectively, were synthesized, and each gene fused with thioredoxin A (TrxA) to construct the expression vectors. The plasmids were transformed into E. coli BL21 (DE3) strains and cultured in MBL medium, which gave high volumetric productivity of HBD5 and HBD6 fusion proteins of up to 1.49 g L(-1) and 1.57 g L(-1), respectively. Soluble HBD5 and HBD6 fusion proteins account for 95.2% and 97.6% of the total fusion proteins, respectively. After cell disruption, the soluble fusion proteins were recovered by affinity chromatography and cleaved by enterokinase. Pure HBD5 and HBD6 were recovered using cationic exchange chromatography. The overall recoveries of HBD5 and HBD6 were 38% and 35%, respectively. Importantly, both HBD5 and HBD6 products showed antimicrobial activity against E. coli but not Staphylococcus aureus. Antimicrobial activity against E. coli of both HBD5 and HBD6 were suppressed by NaCl.
authors.affiliation: School of Chemical and Biomedical Engineering, Nanyang Technological University, 62 Nanyang Drive, Singapore 637459, Singapore.
authors.forename: Lei
authors.initials: L
authors.lastname: Huang
authors.forename: Chi Bun
authors.initials: CB
authors.lastname: Ching
authors.forename: Rongrong
authors.initials: R
authors.lastname: Jiang
authors.forename: Susanna Su Jan
authors.initials: SS
authors.lastname: Leong
mesh_terms: D000900:Anti-Bacterial Agents / Q000096:biosynthesis* / Q000494:pharmacology; D001483:Base Sequence; D002846:Chromatography, Affinity; D004305:Dose-Response Relationship, Drug; D004926:Escherichia coli / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008826:Microbial Sensitivity Tests; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000494:pharmacology; D012995:Solubility; D023083:beta-Defensins / Q000096:biosynthesis* / Q000235:genetics / Q000494:pharmacology
chemical_list: D000900:Anti-Bacterial Agents; C465593:DEFB105A protein, human; C465592:DEFB106A protein, human; D011993:Recombinant Fusion Proteins; D023083:beta-Defensins