Oxidative refolding from inclusion bodies

Christopher A. Nelson; Chung A. Lee; Daved H. Fremont

doi:10.1007/978-1-4939-0354-2_11

Oxidative refolding from inclusion bodies

Christopher A. Nelson, Chung A. Lee, Daved H. Fremont

Research output: Contribution to journal › Article › peer-review

16 Scopus citations

Abstract

This protocol describes the growth and purification of bacterial inclusion body proteins with an option to selenomethionine label the targeted protein through feedback inhibition of methionine biosynthesis in common (non-auxotrophic) strains of E. coli. The method includes solubilization of inclusion body proteins by chemical denaturation and disulfide reduction, renaturation of the solubilized material through rapid dilution by pulsed injection into refolding buffer containing arginine and a mixture of oxidized and reduced glutathione, recovery of the recombinant protein using a stirred cell concentrator, and removal of the aggregated or misfolded fraction by passage over size-exclusion chromatography. The quality of the resulting protein can be assessed by SDS-PAGE.

Original language	English
Pages (from-to)	145-157
Number of pages	13
Journal	Methods in Molecular Biology
Volume	1140
DOIs	https://doi.org/10.1007/978-1-4939-0354-2_11
State	Published - 2014

Keywords

Aggregation
Cell lysis
Escherichia coli
Feedback inhibition
Inclusion bodies
Induction
Protein concentration
Protein purification
Protein refolding
Recombinant protein expression
Selenomethionine labeling
Size-exclusion chromatography
Solubilization

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10.1007/978-1-4939-0354-2_11

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title = "Oxidative refolding from inclusion bodies",

abstract = "This protocol describes the growth and purification of bacterial inclusion body proteins with an option to selenomethionine label the targeted protein through feedback inhibition of methionine biosynthesis in common (non-auxotrophic) strains of E. coli. The method includes solubilization of inclusion body proteins by chemical denaturation and disulfide reduction, renaturation of the solubilized material through rapid dilution by pulsed injection into refolding buffer containing arginine and a mixture of oxidized and reduced glutathione, recovery of the recombinant protein using a stirred cell concentrator, and removal of the aggregated or misfolded fraction by passage over size-exclusion chromatography. The quality of the resulting protein can be assessed by SDS-PAGE.",

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doi = "10.1007/978-1-4939-0354-2_11",

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T1 - Oxidative refolding from inclusion bodies

AU - Nelson, Christopher A.

AU - Lee, Chung A.

AU - Fremont, Daved H.

N1 - Publisher Copyright: © Springer Science+Business Media New York 2014.

PY - 2014

Y1 - 2014

N2 - This protocol describes the growth and purification of bacterial inclusion body proteins with an option to selenomethionine label the targeted protein through feedback inhibition of methionine biosynthesis in common (non-auxotrophic) strains of E. coli. The method includes solubilization of inclusion body proteins by chemical denaturation and disulfide reduction, renaturation of the solubilized material through rapid dilution by pulsed injection into refolding buffer containing arginine and a mixture of oxidized and reduced glutathione, recovery of the recombinant protein using a stirred cell concentrator, and removal of the aggregated or misfolded fraction by passage over size-exclusion chromatography. The quality of the resulting protein can be assessed by SDS-PAGE.

AB - This protocol describes the growth and purification of bacterial inclusion body proteins with an option to selenomethionine label the targeted protein through feedback inhibition of methionine biosynthesis in common (non-auxotrophic) strains of E. coli. The method includes solubilization of inclusion body proteins by chemical denaturation and disulfide reduction, renaturation of the solubilized material through rapid dilution by pulsed injection into refolding buffer containing arginine and a mixture of oxidized and reduced glutathione, recovery of the recombinant protein using a stirred cell concentrator, and removal of the aggregated or misfolded fraction by passage over size-exclusion chromatography. The quality of the resulting protein can be assessed by SDS-PAGE.

KW - Aggregation

KW - Cell lysis

KW - Escherichia coli

KW - Feedback inhibition

KW - Inclusion bodies

KW - Induction

KW - Protein concentration

KW - Protein purification

KW - Protein refolding

KW - Recombinant protein expression

KW - Selenomethionine labeling

KW - Size-exclusion chromatography

KW - Solubilization

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U2 - 10.1007/978-1-4939-0354-2_11

DO - 10.1007/978-1-4939-0354-2_11

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C2 - 24590715

AN - SCOPUS:84907941366

SN - 1064-3745

VL - 1140

SP - 145

EP - 157

JO - Methods in Molecular Biology

JF - Methods in Molecular Biology

ER -

Oxidative refolding from inclusion bodies

Abstract

Keywords

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