title: Expression, purification, and antibacterial activity of bovine lactoferrampin-lactoferricin in Pichia pastoris.
abstract: Bovine lactoferrampin (LFA) and bovine lactoferricin (LFC) are two antimicrobial peptides located in the N(1) domain of bovine lactoferrin. The bactericidal activity of the fused peptide LFA-LFC is stronger than that of either LFA or LFC. The high cost of peptide production from either native digestion or chemical synthesis limits the clinical application of antimicrobial peptides. The expression of recombinant peptides in yeast may be an effective alternative. In the current study, the expression, purification, and antibacterial activity of LFA-LFC using the Pichia pastoris expression system are reported. The linearized expression vector pPICZaA-LFA-LFC was transformed into P. pastoris KM71 by electroporation, and positive colonies harboring the target genes were screened out and used for fermentation. The recombinant LFA-LFC peptide was purified via two-step column chromatography and identified by tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The results indicate that P. pastoris is a suitable system for secreting LFA-LFC. The fermentation supernate and the purified LFA-LFC show high antimicrobial activities. The current study is the first to report on the expression and purification of LFA-LFC in P. pastoris and may have potential practical applications in microbial peptide production.
authors: Research Center for Healthy Breeding of Livestock and Poultry, Institute of Subtropical Agriculture, CAS, Changsha, Hunan 410125, China.
authors: Xiang-Shan
authors: XS
authors: Tang
authors: Zhi-Ru
authors: ZR
authors: Tang
authors: Sheng-Ping
authors: SP
authors: Wang
authors: Ze-Meng
authors: ZM
authors: Feng
authors: Dong
authors: D
authors: Zhou
authors: Tie-Jun
authors: TJ
authors: Li
authors: Yu-Long
authors: YL
authors: Yin
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000900:Anti-Bacterial Agents / Q000737:chemistry / Q000494:pharmacology*; D023181:Antimicrobial Cationic Peptides / Q000235:genetics* / Q000302:isolation & purification / Q000494:pharmacology; D002417:Cattle; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D018274:Electroporation; D004926:Escherichia coli / Q000187:drug effects* / Q000254:growth & development; D007781:Lactoferrin / Q000737:chemistry / Q000235:genetics*; D008826:Microbial Sensitivity Tests; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics*; D010843:Pichia / Q000235:genetics* / Q000378:metabolism; D010957:Plasmids; D011993:Recombinant Fusion Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000494:pharmacology; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014162:Transfection
chemical_list: D000900:Anti-Bacterial Agents; D023181:Antimicrobial Cationic Peptides; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; C502040:lactoferrampin; C082171:lactoferricin B; D007781:Lactoferrin