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

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Title: Expression, purification and characterization of the recombinant cysteine-rich antimicrobial peptide snakin-1 in Pichia pastoris
Authors: Kuddus, Md. Ruhul Browse this author
Rumi, Farhana Browse this author
Tsutsumi, Motosuke Browse this author
Takahashi, Rika Browse this author
Yamano, Megumi Browse this author
Kamiya, Masakatsu Browse this author
Kikukawa, Takashi Browse this author
Demura, Makoto Browse this author
Aizawa, Tomoyasu Browse this author →KAKEN DB
Keywords: Antimicrobial peptide
Cysteine-rich
Snakin-1
Pichia pastoris
Over-expression
Folding
Membrane permeability
Issue Date: Jun-2016
Publisher: Elsevier
Journal Title: Protein expression and purification
Volume: 122
Start Page: 15
End Page: 22
Publisher DOI: 10.1016/j.pep.2016.02.002
PMID: 26854372
Abstract: 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 http://creativecommons.org/licenses/by-nc-nd/4.0/
http://creativecommons.org/licenses/by-nc-nd/4.0/
Type: article (author version)
URI: http://hdl.handle.net/2115/65808
Appears in Collections:生命科学院・先端生命科学研究院 (Graduate School of Life Science / Faculty of Advanced Life Science) > 雑誌発表論文等 (Peer-reviewed Journal Articles, etc)

Submitter: 相沢 智康

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