title: High-level production of a candidacidal peptide lactoferrampin in Escherichia coli by fusion expression.
abstract: Expression of lactoferrampin 265-284 (Lfampin20), a potential candidacidal agent with 20 amino acid segment from lactoferrin, in Escherichia coli was explored. The DNA fragment encoding Lfampin20 was synthesized in light of the E. coli preferred codons by "partially overlapping primer-based PCR" method. The Lfampin20 gene was fused with thioredoxin (Trx) gene to construct a recombinant plasmid pETLfa20. The resulting expression level of the fusion protein Trx-Lfampin20 (approximately 20 kDa) accounted for 34-42% of cellular protein, and about 52% of the target proteins were in a soluble form. Soluble Trx-Lfampin20 accounted for 66% of the total soluble proteins. The soluble fusion protein was easily purified to near homogeneity by affinity chromatography using hexahistidine tag. Recombinant Lfampin20 was effectively obtained by on-column cleavage of the fusion protein with factor Xa. An unknown site in the Trx-tag fusion protein, which can be cleaved by factor Xa to produce approximately 10 kDa protein, was found. Compared with the unknown site, the specific site of IEGR[downwards arrow]X was easier to be recognized and cleaved by factor Xa. The molecular mass of recombinant Lfampin20 determined by MALDI-TOF (matrix assisted laser desorption ionization-time-of-flight) is equal to its theoretical molecular weight. Antimicrobial activity assays demonstrated that the recombinant Lfampin20 had candidacidal activity. Integration of the key strategies for the expression of antimicrobial peptides (AMPs) such as codon usage bias, fusion partner and on-column cleavage, would provide an efficient and facile platform for the production or study of AMPs.
authors.affiliation: Gene Engineering Laboratory, Feed Research Institute, Chinese Academy of Agricultural Sciences, 12 Zhongguancun Nandajie St., Haidian District, Beijing 100081, PR China.
authors.forename: Yalin
authors.initials: Y
authors.lastname: Yanga
authors.forename: Zigang
authors.initials: Z
authors.lastname: Tiana
authors.forename: Da
authors.initials: D
authors.lastname: Teng
authors.forename: Jun
authors.initials: J
authors.lastname: Zhang
authors.forename: Jiarong
authors.initials: J
authors.lastname: Wang
authors.forename: Jianhua
authors.initials: J
authors.lastname: Wang
mesh_terms: D000935:Antifungal Agents / Q000494:pharmacology; D001483:Base Sequence; D002176:Candida albicans / Q000187:drug effects; D002846:Chromatography, Affinity; D004926:Escherichia coli / Q000235:genetics*; D007781:Lactoferrin / Q000096:biosynthesis* / Q000235:genetics / Q000494:pharmacology; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000096:biosynthesis* / Q000235:genetics / Q000494:pharmacology; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000494:pharmacology; D016415:Sequence Alignment; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
chemical_list: D000935:Antifungal Agents; C508417:LFampin 265-284; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; D007781:Lactoferrin