title: Expression of recombinant hybrid peptide cecropinA(1-8)-magainin2(1-12) in Pichia pastoris: purification and characterization.
abstract: Hybrid antibacterial peptide CA-MA (cecropinA(1-8)-magainin2(1-12)) is a linear cationic peptide that has potent antimicrobial properties without hemolytic activity. To explore a new approach of expression of hybrid peptide CA-MA in methylotrophic yeast, Pichia pastoris, the gene of CA-MA was obtained by recursive PCR (rPCR) and cloned into the vector pPICZalpha-A. The SalI-linearized plasmid pPICZalpha-CA-MA was transformed into P. pastoris SMD1168 by electroporation. The expression was induced for 96h with 1.0% methanol at 28 degrees C, pH 5.0. Recombinant CA-MA was purified by reversed-phase HPLC and 22 mg pure active CA-MA was obtained from 1L fermentation culture. Tricine-SDS-PAGE indicated that recombinant CA-MA protein molecular weight is 2.6 kDa. Mass spectrometry of purified CA-MA demonstrated a single large signal for the molecular ion [M+2H+](2+) at 1281.07 m/z, identical to that of the putative protein (2.56 kDa). Antimicrobial assays showed that CA-MA has a broad spectrum of antimicrobial property against fungi, as well as Gram-positive and Gram-negative bacteria. This is the first report on the heterologous expression of a hybrid antibacterial peptide with molecular weight below 3.0 kDa in P. pastoris. Our results demonstrate that functional CA-MA can be produced in sufficient quantities using P. pastoris for use in further studies on functionality and diagnostic applications.
authors.affiliation: State Key Laboratory of Biocontrol, College of Life Sciences, Sun Yat-sen (Zhongshan) University, Guangzhou 510275, PR China.
authors.forename: Fengliang
authors.initials: F
authors.lastname: Jin
authors.forename: Xiaoxia
authors.initials: X
authors.lastname: Xu
authors.forename: Liexi
authors.initials: L
authors.lastname: Wang
authors.forename: Wenqing
authors.initials: W
authors.lastname: Zhang
authors.forename: Dexiang
authors.initials: D
authors.lastname: Gu
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000890:Anti-Infective Agents / Q000302:isolation & purification* / Q000494:pharmacology; D023181:Antimicrobial Cationic Peptides / Q000235:genetics*; D001483:Base Sequence; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D005658:Fungi / Q000187:drug effects; D006090:Gram-Negative Bacteria / Q000187:drug effects; D006094:Gram-Positive Bacteria / Q000187:drug effects; D006461:Hemolysis; D054806:Magainins; D051379:Mice; D008969:Molecular Sequence Data; D010843:Pichia / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000494:pharmacology; D021241:Spectrometry, Mass, Electrospray Ionization; D029867:Xenopus Proteins / Q000235:genetics*
chemical_list: D000890:Anti-Infective Agents; D023181:Antimicrobial Cationic Peptides; D054806:Magainins; D011993:Recombinant Fusion Proteins; D029867:Xenopus Proteins; C053301:magainin 2 peptide, Xenopus; C031161:cecropin A