title: Molecular cloning, expression and purification of lactoferrin from Tibetan sheep mammary gland using a yeast expression system.
abstract: This paper reports the successful expression of a lactoferrin gene-obtained from the mammary gland tissue of Tibetan sheep-in the yeast Pichia pastoris GS115 using pPICZαA as the recombinant plasmid and α-factor signal sequence for secretion. The recombinant lactoferrin was purified by ammonium sulfate precipitation, ion-exchange column chromatography and gel-filtration chromatography, and it had a molecular mass of 76kDa. We obtained an expression yield of >60mgL(-1) and specific activity of 2533.33Umg(-1). The antimicrobial activities and iron-binding behaviors of recombinant lactoferrin indicated that it was correctly folded and functional. Additionally, recombinant lactoferrin inhibited the growth of Escherichia coli JM109 and Staphylococcus aureus. These findings indicate that recombinant lactoferrin is a potential antibiotic for use on humans. This study also demonstrates the successful expression of recombinant lactoferrin using the eukaryotic host organism P. pastoris, paving the way for protein engineering using this gene.
authors: Key Laboratory of Sichuan Province for Qinghai-Tibetan Plateau Animal Genetic Resource Reservation and Exploitation, Chengdu 610041, PR China; College of Life Science and Technology, Southwest University for Nationalities, Chengdu 610041, PR China.
authors: Jianbo
authors: J
authors: Li
authors: Key Laboratory of Sichuan Province for Qinghai-Tibetan Plateau Animal Genetic Resource Reservation and Exploitation, Chengdu 610041, PR China; College of Life Science and Technology, Southwest University for Nationalities, Chengdu 610041, PR China.
authors: Wuzheng
authors: W
authors: Zhu
authors: College of Life Science and Technology, Southwest University for Nationalities, Chengdu 610041, PR China.
authors: Meirong
authors: M
authors: Luo
authors: College of Life Science and Technology, Southwest University for Nationalities, Chengdu 610041, PR China.
authors: Honghui
authors: H
authors: Ren
authors: College of Life Science and Technology, Southwest University for Nationalities, Chengdu 610041, PR China.
authors: Lu
authors: L
authors: Tang
authors: College of Life Science and Technology, Southwest University for Nationalities, Chengdu 610041, PR China.
authors: Honghai
authors: H
authors: Liao
authors: Key Laboratory of Sichuan Province for Qinghai-Tibetan Plateau Animal Genetic Resource Reservation and Exploitation, Chengdu 610041, PR China. Electronic address: wangyong010101@swun.cn.
authors: Yong
authors: Y
authors: Wang
mesh_terms: D000818:Animals; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D003001:Cloning, Molecular / Q000379:methods*; D018076:DNA, Complementary / Q000235:genetics; D004587:Electrophoresis, Agar Gel; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D015870:Gene Expression; D006863:Hydrogen-Ion Concentration; D007501:Iron / Q000378:metabolism; D007781:Lactoferrin / Q000302:isolation & purification* / Q000378:metabolism* / Q000494:pharmacology; D008321:Mammary Glands, Animal / Q000378:metabolism*; D008826:Microbial Sensitivity Tests; D011485:Protein Binding / Q000187:drug effects; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000494:pharmacology; D012441:Saccharomyces cerevisiae / Q000378:metabolism*; D017422:Sequence Analysis, DNA; D012756:Sheep; D013211:Staphylococcus aureus / Q000187:drug effects / Q000254:growth & development
chemical_list: D000900:Anti-Bacterial Agents; D018076:DNA, Complementary; D011994:Recombinant Proteins; D007501:Iron; D007781:Lactoferrin
keywords: Antimicrobial activity; Iron-binding behavior; Pichia pastoris; Purification; Recombinant lactoferrin; Tibetan sheep