title: Production of recombinant porcine lactoferrin exhibiting antibacterial activity in methylotrophic yeast, Pichia pastoris.
abstract: Lactoferrin is a metal-binding glycoprotein exhibiting multifunctional immunoregulation of antibacterial, antioxidant, anti-endotoxin and antiviral activities. Uptake of porcine lactoferrin (PLF) has been shown to enhance resistance to diarrhea and anemia in neonatal piglets. In this study, the methylotrophic yeast, Pichia pastoris, was used to express a recombinant PLF (rPLF) gene from swine mammary gland. A synthetic secretion cassette was constructed using the inducible promoter of the alcohol oxidase-1 gene (AOX1) and the yeast alpha-mating factor signal peptide. After electroporation and Zeocin selection, several clones expressed high levels of rPLF protein which constitutes more than 30% of the total protein. A time-course study showed that rPLF mRNA transcripts are stably expressed during 120 h of culture induction. rPLF was exported into the culture supernatant at approximately 87 mg/l and a large portion of rPLF was accumulated in the cell cytoplasm at approximately 760 mg/l after 72 h of methanol induction. Recombinant PLF protein was purified via a heparin column using a fast protein liquid chromatography system. The glycosylation of P. pastoris-derived rPLF was analyzed and similar patterns to milk PLF were observed. Pepsin hydrolysate of rPLF displayed high bactericidal activity against Escherichia coli ATCC 25922 under scanning electron microscopy observation and minimal inhibitory concentration and minimal bactericidal concentration tests. Our results suggested that the methylotrophic yeast-inducible system is suitable for large-scale production of active antibacterial rPLF glycoprotein.
authors.affiliation: Department of Molecular Biotechnology, Da-Yeh University, Changhwa, Taiwan.
authors.forename: Hsiao-Ling
authors.initials: HL
authors.lastname: Chen
authors.forename: Yi-Wen
authors.initials: YW
authors.lastname: Lai
authors.forename: Chih-Ching
authors.initials: CC
authors.lastname: Yen
authors.forename: Yuh-Yih
authors.initials: YY
authors.lastname: Lin
authors.forename: Chien-Yu
authors.initials: CY
authors.lastname: Lu
authors.forename: Shang-Hsun
authors.initials: SH
authors.lastname: Yang
authors.forename: Tung-Chou
authors.initials: TC
authors.lastname: Tsai
authors.forename: Yi-Jiun
authors.initials: YJ
authors.lastname: Lin
authors.forename: Chin-Wen
authors.initials: CW
authors.lastname: Lin
authors.forename: Chuan-Mu
authors.initials: CM
authors.lastname: Chen
mesh_terms: D000818:Animals; D000900:Anti-Bacterial Agents / Q000302:isolation & purification / Q000494:pharmacology*; D001483:Base Sequence; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000187:drug effects; D007781:Lactoferrin / Q000235:genetics / Q000302:isolation & purification / Q000494:pharmacology*; D008826:Microbial Sensitivity Tests; D008855:Microscopy, Electron, Scanning; D008969:Molecular Sequence Data; D010455:Peptides / Q000235:genetics / Q000302:isolation & purification / Q000494:pharmacology; D010843:Pichia / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000494:pharmacology; D013552:Swine
chemical_list: D000900:Anti-Bacterial Agents; D010455:Peptides; D011994:Recombinant Proteins; D007781:Lactoferrin