Recombinant human retinol-binding protein refolding, native disulfide formation, and characterization

Yansheng Xie, Hilal A. Lashuel, Greta J. Miroy, Sergei Dikler, Jeffery W. Kelly

Research output: Contribution to journalArticle

26 Citations (Scopus)

Abstract

Human retinol-binding protein (RBP) is a monomeric 21-kDa protein that is currently the subject of numerous studies owing to its role in the cellular uptake and utilization of retinol. When the RBP gene is overexpressed in Escherichia coli, inclusion bodies of aggregated RBP are found in the cells. These inclusion bodies are solubilized in 5.0 M GdmCl containing 10 mM DTT. Refolding of RBP is carried out in the presence of vitamin A by diluting denatured and reduced RBP into a redox refolding buffer consisting of 3 mM cysteine/0.3 mM cystine at 4°C. Ion exchange chromatography (HPLC) is utilized to purify refolded RBP to homogeneity as demonstrated by SDS-PAGE and electrospray MS. The native structure of refolded RBP was established by its ability to bind to vitamin A and the plasma protein transthyretin. The reconstitution of RBP outlined within affords a 50-60% overall yield, i.e., 73 mg of pure RBP/L of E. coli culture.

Original languageEnglish
Pages (from-to)31-37
Number of pages7
JournalProtein Expression and Purification
Volume14
Issue number1
DOIs
Publication statusPublished - 1 Oct 1998
Externally publishedYes

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Protein Refolding
Retinol-Binding Proteins
Disulfides
Vitamin A
Inclusion Bodies
Escherichia coli
Prealbumin
Cystine
Ion Exchange Chromatography
Chromatography
Oxidation-Reduction
Cysteine
Blood Proteins
Polyacrylamide Gel Electrophoresis
Ion exchange
Buffers
Genes
High Pressure Liquid Chromatography

ASJC Scopus subject areas

  • Biochemistry

Cite this

Recombinant human retinol-binding protein refolding, native disulfide formation, and characterization. / Xie, Yansheng; Lashuel, Hilal A.; Miroy, Greta J.; Dikler, Sergei; Kelly, Jeffery W.

In: Protein Expression and Purification, Vol. 14, No. 1, 01.10.1998, p. 31-37.

Research output: Contribution to journalArticle

Xie, Yansheng ; Lashuel, Hilal A. ; Miroy, Greta J. ; Dikler, Sergei ; Kelly, Jeffery W. / Recombinant human retinol-binding protein refolding, native disulfide formation, and characterization. In: Protein Expression and Purification. 1998 ; Vol. 14, No. 1. pp. 31-37.
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