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Expression, purification and characterization of the recombinant cysteine-rich antimicrobial peptide snakin-1 in Pichia pastoris


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タイトル: Expression, purification and characterization of the recombinant cysteine-rich antimicrobial peptide snakin-1 in Pichia pastoris
著者: Kuddus, Md. Ruhul 著作を一覧する
Rumi, Farhana 著作を一覧する
Tsutsumi, Motosuke 著作を一覧する
Takahashi, Rika 著作を一覧する
Yamano, Megumi 著作を一覧する
Kamiya, Masakatsu 著作を一覧する
Kikukawa, Takashi 著作を一覧する
Demura, Makoto 著作を一覧する
Aizawa, Tomoyasu 著作を一覧する
キーワード: Antimicrobial peptide
Pichia pastoris
Membrane permeability
発行日: 2016年 6月
出版者: Elsevier
誌名: Protein expression and purification
巻: 122
開始ページ: 15
終了ページ: 22
出版社 DOI: 10.1016/j.pep.2016.02.002
抄録: Snakin-1 (SN-1) is a small cysteine-rich plant antimicrobial peptide with broad spectrum antimicrobial activity which was isolated from potato (Solanum tuberosum). Here, we carried out the expression of a recombinant SN-1 in the methylotrophic yeast Pichia pastoris, along with its purification and characterization. A DNA fragment encoding the mature SN-1 was cloned into pPIC9 vector and introduced into P. pastoris. A large amount of pure recombinant SN-1 (approximately 40 mg/1L culture) was obtained from a fed-batch fermentation culture after purification with a cation exchange column followed by RP-HPLC. The identity of the recombinant SN-1 was verified by MALDI-TOF MS, CD and H-1 NMR experiments. All these data strongly indicated that the recombinant SN-1 peptide had a folding with six disulfide bonds that was identical to the native SN-1. Our findings showed that SN-1 exhibited strong antimicrobial activity against test microorganisms and produced very weak hemolysis of mammalian erythrocytes. The mechanism of its antimicrobial action against Escherichia coli was investigated by both outer membrane permeability assay and cytoplasmic membrane depolarization assay. These assays demonstrated that SN-1 is a membrane-active antimicrobial peptide which can disrupt both outer and cytoplasmic membrane integrity. This is the first report on the recombinant expression and purification of a fully active SN-1 in P. pastoris. (C) 2016 Elsevier Inc. All rights reserved.
Rights: © 2016, Elsevier. Licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International
資料タイプ: article (author version)
出現コレクション:雑誌発表論文等 (Peer-reviewed Journal Articles, etc)

提供者: 相沢 智康


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