Recombinant human retinol-binding protein refolding, native disulfide formation, and characterization.
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 degreesC. 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.
Department of Chemistry and the Skaggs Institute of Chemical Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, MB12, La Jolla, California, 92037, USA.
Y
Y
Xie
H A
HA
Lashuel
G J
GJ
Miroy
S
S
Dikler
J W
JW
Kelly
D002852:Chromatography, Ion Exchange; D004220:Disulfides / Q000737:chemistry; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D006801:Humans; D066298:In Vitro Techniques; D002479:Inclusion Bodies / Q000737:chemistry; D017510:Protein Folding; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012177:Retinol-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D054839:Retinol-Binding Proteins, Plasma; D012995:Solubility; D014801:Vitamin A / Q000378:metabolism
D004220:Disulfides; D011994:Recombinant Proteins; D012177:Retinol-Binding Proteins; D054839:Retinol-Binding Proteins, Plasma; D014801:Vitamin A