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Glutathione Ethylester, a Novel Protein Refolding Reagent, Enhances both the Efficiency of Refolding and Correct Disulfide Formation

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Abstract

Protein refolding constitutes a crucial process for recombinant proteins. We report here on the development of a multifunctional refolding additive, glutathione ethyl ester (GSHEE), prepared from a redox reagent glutathione and an amino acid ethyl ester, an aggregation suppressor. Compared to glutathione, GSHEE showed 3.2-fold higher efficiency for the refolding yield of hen egg lysozyme. More importantly, a low concentration of GSHEE is more effective for refolding than conventional additives, such as amino acid ethyl esters by two orders of magnitude. The high potency of GSHEE makes it a candidate for use as a refolding additive for use in conjunction with reduced and denatured proteins.

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Abbreviations

GSH:

Reduced glutathione

GSSG:

Oxidized glutathione

GSHEE:

Reduced glutathione ethyl ester

GSSGEE:

Oxidized glutathione ethyl ester

p-TsOH:

para-Toluene sulfonic acid

BF3·OEt2 :

Boron trifluoride diethyl etherate

(Boc)2O:

Di-t-butyl dicarbonate

Ph:

Phenyl

Et:

Ethyl

AcOH:

Acetic acid

TFA:

Trifluoroacetic acid

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Authors and Affiliations

  1. Japan Synchrotron Radiation Research Institute (SPring-8), 1-1-1 Kouto, Sayo, 679-5198, Hyogo, Japan

    Len Ito & Takashi Kumasaka

  2. School of Science and Technology, Kwansei Gakuin University, Sanda, 669-1337, Hyogo, Japan

    Len Ito, Masaki Okumura, Kohsaku Tao, Akiko Oosuka, Hidetoshi Yamada, Tomohisa Shibano & Hiroshi Yamaguchi

  3. Milbon Co., Ltd., 2-3-35 Zengenzi, Miyakozima, 534-0015, Osaka, Japan

    Len Ito

  4. Laboratory of Biostructural Chemistry, Graduate School of Life Sciences, Tohoku University, Sendai, 980-8577, Miyagi, Japan

    Yusuke Kasai

  5. Institute of Applied Physics, University of Tsukuba, Tsukuba, 305-8573, Ibaraki, Japan

    Shunsuke Tomita & Kentaro Shiraki

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  1. Len Ito
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  2. Masaki Okumura
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  3. Kohsaku Tao
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  9. Kentaro Shiraki
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  10. Takashi Kumasaka
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  11. Hiroshi Yamaguchi
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Correspondence to Len Ito.

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Ito, L., Okumura, M., Tao, K. et al. Glutathione Ethylester, a Novel Protein Refolding Reagent, Enhances both the Efficiency of Refolding and Correct Disulfide Formation. Protein J 31, 499–503 (2012). https://doi.org/10.1007/s10930-012-9427-4

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