title: [Expression and purification of the antimicrobial polypeptide by fusion with elastin-like polypeptide].
abstract: Elastin-like polypeptides (ELPs) are biological macromolecules designed on the elastic structure and composition. ELPs are thermally responsive polypeptides that undergo reversible inverse phase transition. Below their inverse transition temperature (Tt), ELPs are soluble in water, but when the temperature is raised above Tt, phase transition occurs, leading to aggregation of the polypeptide. Based on this property of ELPs, we investigated the ELPs as a fusion partner for the expression and purification of the antimicrobial polypeptide halocidin18 (18-amino-acid subunit, Hal18). According to the amino acid sequence of hal18 and the bias for preferred codons of E. coli, the hal18 gene was synthesized by reverse PCR method and fused with the ELPs gene; the fusion gene was cloned into the prokaryotic expression vector pET23a to construct a recombinant expression vector pETH9E and then transformed into E. coli BL21 (DE3) to overexpress the recombinant fusion protein H9E. H9E was purified by inverse transition cycling (ITC) from the soluble fraction of the lysed E. coli. SDS-PAGE analysis results demonstrated that one-round of ITC purification could remove most of the contaminated proteins, and second-round could increase the purity of the fusion protein up to 95%. After cleavage and dialysis, Hal18 showed strong antimicrobial activities against E. coli and M. luteus which were used as representative gram-negative and gram-positive bacteria, respectively. The results demonstrate that ELPs is an ideal fusion protein to express and purify the antimicrobial polypeptides.
authors.affiliation: College of Life Science, Hubei University, Wuhan.
authors.forename: Fan
authors.initials: F
authors.lastname: Hu
authors.forename: Tao
authors.initials: T
authors.lastname: Ke
authors.forename: Xin
authors.initials: X
authors.lastname: Li
authors.forename: Feng Li
authors.initials: FL
authors.lastname: Hui
authors.forename: Li
authors.initials: L
authors.lastname: Xu
authors.forename: Xiang Dong
authors.initials: XD
authors.lastname: Ma
authors.forename: Li Xin
authors.initials: LX
authors.lastname: Ma
mesh_terms: D000900:Anti-Bacterial Agents / Q000302:isolation & purification* / Q000378:metabolism / Q000494:pharmacology; D023181:Antimicrobial Cationic Peptides / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism / Q000494:pharmacology; D001419:Bacteria / Q000187:drug effects; D004549:Elastin / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism
chemical_list: D000900:Anti-Bacterial Agents; D023181:Antimicrobial Cationic Peptides; D011993:Recombinant Fusion Proteins; D004549:Elastin