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Efficient production of a correctly folded mouse α-defensin, cryptdin-4, by refolding during inclusion body solubilization

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Title: Efficient production of a correctly folded mouse α-defensin, cryptdin-4, by refolding during inclusion body solubilization
Authors: Tomisawa, Satoshi Browse this author
Sato, Yuji Browse this author
Kamiya, Masakatsu Browse this author
Kumaki, Yasuhiro Browse this author
Kikukawa, Takashi Browse this author
Kawano, Keiichi Browse this author
Demura, Makoto Browse this author
Nakamura, Kiminori Browse this author
Ayabe, Tokiyoshi Browse this author
Aizawa, Tomoyasu Browse this author →KAKEN DB
Keywords: α-Defensin
Coexpression
Inclusion bodies
Refolding
NMR
Issue Date: Aug-2015
Publisher: Elsevier
Journal Title: Protein expression and purification
Volume: 112
Start Page: 21
End Page: 28
Publisher DOI: 10.1016/j.pep.2015.04.007
PMID: 25913370
Abstract: Mammalian α-defensins contribute to innate immunity by exerting antimicrobial activity against various pathogens. To perform structural and functional analysis of α-defensins, large amounts of α-defensins are essential. Although many expression systems for the production of recombinant α-defensins have been developed, attempts to obtain large amounts of α-defensins have been only moderately successful. Therefore, in this study, we applied a previously developed aggregation-prone protein coexpression method for the production of mouse α-defensin cryptdin-4 (Crp4) in order to enhance the formation of inclusion bodies in Escherichia coil expression system. By using this method, we succeeded in obtaining a large amount of Crp4 in the form of inclusion bodies. Moreover, we attempted to refold Crp4 directly during the inclusion-body solubilization step under oxidative conditions. Surprisingly, even without any purification, Crp4 was efficiently refolded during the solubilization step of inclusion bodies, and the yield was better than that of the conventional refolding method. NMR spectra of purified Crp4 suggested that it was folded into its correct tertiary structure. Therefore, the method described in this study not only enhances the expression of α-defensin as inclusion bodies, but also eliminates the cumbersome and time-consuming refolding step.
Rights: ©2015, 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/2115/62595
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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