A eukaryotic expression strategy for producing the novel antimicrobial peptide PRW4
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BIOTECHNOLOGY AND INDUSTRIAL MICROBIOLOGY - RESEARCH PAPER
A eukaryotic expression strategy for producing the novel antimicrobial peptide PRW4 Na Zhan 1 & Tianyu Wang 1 & Licong Zhang 1 & Anshan Shan 1 Received: 25 November 2019 / Accepted: 30 April 2020 # Sociedade Brasileira de Microbiologia 2020
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. Keywords Antimicrobial peptides . PRW4 . Pichia pastoris . Antimicrobial activity . Gene expression
Introduction For decades, edible antibiotics have been used in the livestock industry. However, the long-term use of antibiotics, as well as the abuse of these compounds, has contributed to the increase in bacterial resistance, threatening public health [1, 2]. Therefore, it is necessary to find new types of antibiotic substitutes that can be applied to livestock production [3]. In recent decades, antimicrobial peptides (AMPs) have drawn increasing attention due to their rapid and broadspectrum activities against gram-negative and gram-positive Na Zhan and Tianyu Wang contributed to the work equally and should be regarded as co-first authors. Responsible Editor: Gisele Monteiro. Electronic supplementary material The online version of this article (https://doi.org/10.1007/s42770-020-00291-4) contains supplementary material, which is available to authorized users. * Anshan Shan [email protected] 1
Institute of Animal Nutrition, Northeast Agricultural University, No. 600 Changjiang Road, Xiangfang District, Harbin, China
bacteria, fungi, viruses, etc. In addition, the antimicrobial mechanism of AMPs is different from that of antibiotics. AMPs physically destroy the cell membrane structure of bacteria and leak the contents of cells to kill cells, so it is difficult to acquire drug resistance [4–6]. Therefore, AMPs are
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