Renaturation of Escherichia coli-derived recombinant human macrophage colony-stimulating factor.
Recombinant human macrophage colony-stimulating factor (rhM-CSF), a homodimeric, disulfide bonded protein, was expressed in Escherichia coli in the form of inclusion bodies. Reduced and denatured rhM-CSF monomers were refolded in the presence of a thiol mixture (reduced and oxidized glutathione) and a low concentration of denaturing agent (urea or guanidinium chloride). Refolding was monitored by nonreducing gel electrophoresis and recovery of bioactivity. The effects of denaturant type and concentration, protein concentration, concentration of thiol/disulfide reagents, temperature, and presence of impurities on the kinetics of rhM-CSF renaturation were investigated. Low denaturant concentrations (<0.5 M urea) and high protein concentrations (>0.4 mg/ml) in the refolding mixture resulted in increased formation of aggregates, although aggregation was never significant even when refolding was carried out at room temperature. Higher protein concentration resulted in higher rates but did not lead to increased yields, due to the formation of unwanted aggregates. Experiments conducted at room temperature resulted in slightly higher rates than those conducted at 4 degrees C. Although the initial renaturation rate for solubilized inclusion body protein without purification was higher than that of the reversed-phase purified reduced denatured rhM-CSF, the final renaturation yield was much higher for the purified material. A maximum refolding yield of 95% was obtained for the purified material at the following refolding conditions: 0.5 M urea, 50 mM Tris, 1.25 mM DTT, 2 mM GSH, 2 mM GSSG, 22 degrees C, pH 8, [protein] = 0.13 mg/ml.
Tran-Moseman
A
A
Department of Chemical Engineering, Tufts University, Medford, Massachusetts 02155, USA.
Schauer
N
N
De Bernardez Clark
E
E
D019281:Dimerization; D004926:Escherichia coli / Q000235:genetics; D019791:Guanidine; D006801:Humans; D066298:In Vitro Techniques; D007700:Kinetics; D016173:Macrophage Colony-Stimulating Factor / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D010084:Oxidation-Reduction; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D013438:Sulfhydryl Compounds; D013696:Temperature; D014508:Urea
D011994:Recombinant Proteins; D013438:Sulfhydryl Compounds; D016173:Macrophage Colony-Stimulating Factor; D014508:Urea; D019791:Guanidine