A eukaryotic expression strategy for producing the novel antimicrobial peptide PRW4
Na Zhan1, Tianyu Wang1, Licong Zhang1
1Institute of Animal Nutrition, Northeast Agricultural University, No. 600 Changjiang Road, Xiangfang District, Harbin, China.
Abstract:
The antimicrobial peptide PMAP-36 is a cationic peptide derived from porcine myeloid. The N-terminally paired lysine of PMAP-36 was substituted with tryptophan, and the C-terminal hydrophobic tail was deleted, thereby obtaining the antimicrobial peptide PRW4. PRW4 is a α-helical antimicrobial peptide with broad-spectrum antimicrobial activity. In this study, PRW4 was fused to the 6× His-Trx, and the fusion protein was successfully expressed in Pichia pastoris GS115 from the vector pPICZαA. The maximal induction of recombinant protein occurred in the presence of 1% methanol after 96 h at pH 6.0. After purification by a Ni-NTA resin column and digestion by enterokinase protease, 15 mg of recombinant PRW4 with a purity of 90% was obtained from 1 L of fermentation culture. The results indicated that recombinant PRW4 had similar antimicrobial activity as synthetic PRW4 against bacteria such as Escherichia coli ATCC 25922, Escherichia coli UB 1005, Salmonella typhimurium C7731, Salmonella typhimurium 7913, Salmonella typhimurium ATCC 14028, Staphylococcus aureus ATCC 29213, Staphylococcus epidermidis ATCC 12228, and Streptococcus faecalis ATCC 29212. We have successfully expressed PRW4 in P. pastoris, and this work provides a reference for the production of modified antimicrobial peptides in P. pastoris.
Insights
Recombinant antimicrobial peptide PRW4, derived from PMAP-36, was successfully produced in Pichia pastoris. This method yielded a highly pure protein with broad-spectrum antimicrobial activity comparable to its synthetic form.
Area of Science:
- Biotechnology
- Microbiology
- Protein Engineering
Background:
- Antimicrobial peptides (AMPs) are crucial in innate immunity.
- PMAP-36, a porcine myeloid-derived AMP, was modified to create PRW4.
- PRW4 exhibits α-helical structure and broad-spectrum antimicrobial activity.
Purpose of the Study:
- To express and purify a recombinant form of the modified antimicrobial peptide PRW4.
- To evaluate the antimicrobial activity of the recombinant PRW4.
- To establish a production system for modified AMPs in Pichia pastoris.
Main Methods:
- PRW4 was fused to a 6× His-Trx tag for expression.
- Recombinant protein expression was carried out in Pichia pastoris GS115 using the pPICZαA vector.
- Maximal protein induction was achieved at 1% methanol, 96h, pH 6.0.
- Purification involved Ni-NTA resin chromatography and enterokinase digestion.
Main Results:
- Successfully expressed and purified recombinant PRW4 with 90% purity.
- Obtained 15 mg of recombinant PRW4 per liter of fermentation culture.
- Recombinant PRW4 demonstrated antimicrobial activity against a panel of Gram-negative and Gram-positive bacteria, similar to synthetic PRW4.
Conclusions:
- Pichia pastoris is an effective host for the production of recombinant PRW4.
- The recombinant production method provides a viable alternative to synthetic peptide production.
- This study serves as a reference for producing other modified antimicrobial peptides.
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