On-column refolding of corticotropin-releasing factor receptor 1 extracellular domain by size exclusion chromatography.
Expressing the extracellular domain of corticotropin releasing factor receptor 1 in Escherichia coli usually results in the formation of inclusion bodies. Here we describe the optimization of refolding by applying size exclusion chromatography with a denaturing guanidine hydrochloride gradient and a refolding buffer containing glycerol. Several chromatographic parameters like gradient length, flow rate, sample concentration and chromatography resin characteristics were evaluated. Recovery yields of refolded protein above 50% using a Superdex 200 column demonstrate the usefulness of this method.
Ersoy
Maria
M
Global Protein Science and Supply, AstraZeneca R&D Södertälje, SE-15185 Södertälje, Sweden. Stefan.Schmidt@astrazeneca.com
Matic
Sandra
S
Schmidt
Stefan R
SR
D002850:Chromatography, Gel / Q000379:methods*; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D005990:Glycerol / Q000737:chemistry; D019791:Guanidine / Q000737:chemistry; D006801:Humans; D002479:Inclusion Bodies / Q000737:chemistry; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D018019:Receptors, Corticotropin-Releasing Hormone / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D000099052:CRF Receptor, Type 1
D018019:Receptors, Corticotropin-Releasing Hormone; D011994:Recombinant Proteins; D000099052:CRF Receptor, Type 1; D019791:Guanidine; D005990:Glycerol