1. Academic Validation
  2. Characterization of bioactive recombinant antimicrobial peptide parasin I fused with human lysozyme expressed in the yeast Pichia pastoris system

Characterization of bioactive recombinant antimicrobial peptide parasin I fused with human lysozyme expressed in the yeast Pichia pastoris system

  • Enzyme Microb Technol. 2015 Sep;77:61-7. doi: 10.1016/j.enzmictec.2015.06.001.
Hua Zhao 1 Jiayong Tang 2 Lei Cao 2 Gang Jia 2 Dingbiao Long 3 Guangmang Liu 2 Xiaoling Chen 2 Jingyi Cai 2 Haiying Shang 2
Affiliations

Affiliations

  • 1 Animal Nutrition Institute, Sichuan Agricultural University, Chengdu, Sichuan 611130, China. Electronic address: zhua666@126.com.
  • 2 Animal Nutrition Institute, Sichuan Agricultural University, Chengdu, Sichuan 611130, China.
  • 3 Chongqing Academy of Animal Science, Chongqing, 402460, China.
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.

Keywords

Antimicrobial peptide; Bioactive; Fusion protein; Human lysozyme; Parasin I; Pichia pastoris.

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