title: Characterization of bioactive recombinant antimicrobial peptide parasin I fused with human lysozyme expressed in the yeast Pichia pastoris system.
abstract: Parasin I (PI) is a 19 amino acid peptide with potent antimicrobial activities against a broad spectrum of microorganisms and is a good candidate for development as a novel antimicrobial agent. The objective of this study was to express and characterize a codon optimized parasin I peptide fused with human lysozyme (hLY). A 513 bp cDNA fragment encoding the mature hLY protein and parasin I peptide was designed and synthesized according to the codon bias of Pichia pastoris. A 4×Gly flexible amino acid linker with an enterokinase cleavage (DDDDK) was designed to link the PI to the C-terminal of hLY. The codon optimized recombinant hLY-PI was cloned into the pPICZαA vector and expressed in P. pastoris. The over-expressed extracellular rehLY-PI was purified using Ni sepharose affinity column and exhibited a molecular mass of approximately 18 kDa. After digested with enterokinase the rehLY-PI protein release its corresponding rehLY and rePI, with molecular mass of 16 kDa and 2 kDa, respectively, on Tricine-SDS-PAGE. The released rehLY exhibited similar lytical activity against Micrococcus lysodeikticus to its commercial hLY. The digested rehLY-PI product exhibited antimicrobial activities against Bacillus subtilis, Staphylococcus aureus and Escherichia coli, and synergism has been found between the released rePI and rehLY. In conclusion, we successfully optimized a rehLY-PI fusion protein encoding gene and over-expressed the rehLY-PI in P. pastoris. The recombination protein digested with enterokinase released functional hLY and antimicrobial parasin I, which demonstrates a potential for future use as an animal feed additive to partly replace antibiotic.
authors.affiliation: Animal Nutrition Institute, Sichuan Agricultural University, Chengdu, Sichuan 611130, China. Electronic address: zhua666@126.com.
authors.forename: Hua
authors.initials: H
authors.lastname: Zhao
authors.affiliation: Animal Nutrition Institute, Sichuan Agricultural University, Chengdu, Sichuan 611130, China.
authors.forename: Jiayong
authors.initials: J
authors.lastname: Tang
authors.affiliation: Animal Nutrition Institute, Sichuan Agricultural University, Chengdu, Sichuan 611130, China.
authors.forename: Lei
authors.initials: L
authors.lastname: Cao
authors.affiliation: Animal Nutrition Institute, Sichuan Agricultural University, Chengdu, Sichuan 611130, China.
authors.forename: Gang
authors.initials: G
authors.lastname: Jia
authors.affiliation: Chongqing Academy of Animal Science, Chongqing, 402460, China.
authors.forename: Dingbiao
authors.initials: D
authors.lastname: Long
authors.affiliation: Animal Nutrition Institute, Sichuan Agricultural University, Chengdu, Sichuan 611130, China.
authors.forename: Guangmang
authors.initials: G
authors.lastname: Liu
authors.affiliation: Animal Nutrition Institute, Sichuan Agricultural University, Chengdu, Sichuan 611130, China.
authors.forename: Xiaoling
authors.initials: X
authors.lastname: Chen
authors.affiliation: Animal Nutrition Institute, Sichuan Agricultural University, Chengdu, Sichuan 611130, China.
authors.forename: Jingyi
authors.initials: J
authors.lastname: Cai
authors.affiliation: Animal Nutrition Institute, Sichuan Agricultural University, Chengdu, Sichuan 611130, China.
authors.forename: Haiying
authors.initials: H
authors.lastname: Shang
mesh_terms: D000818:Animals; D000890:Anti-Infective Agents / Q000494:pharmacology; D023181:Antimicrobial Cationic Peptides / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D001412:Bacillus subtilis / Q000187:drug effects; D004926:Escherichia coli / Q000187:drug effects; D006801:Humans; D009113:Muramidase / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D010843:Pichia / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology; D013211:Staphylococcus aureus / Q000187:drug effects
chemical_list: D000890:Anti-Infective Agents; D023181:Antimicrobial Cationic Peptides; D011993:Recombinant Fusion Proteins; C115701:parasin I; D009113:Muramidase
keywords: Antimicrobial peptide; Bioactive; Fusion protein; Human lysozyme; Parasin I; Pichia pastoris