Refolding and single-step purification of porcine interferon-gamma from Escherichia coli inclusion bodies. Conditions for reconstitution of dimeric IFN-gamma.
Recombinant porcine interferon-gamma, overexpressed in Escherichia coli, was found to accumulate in cytoplasmic inclusion bodies. The influence of various physicochemical parameters on refolding was investigated using 6 M guanidine/HCl-solubilised inclusion bodies which had been purified by ultracentrifugation on a sucrose step gradient. It appeared that the yield of reconstitution of denatured protein reached 60-70% under optimum conditions, i.e. at an intermediary guanidine/HCl concentration of 0.5 M and at a protein concentration of 10-20 microM (0 degrees C). Since intermediary guanidine/HCl concentrations at 0.5-1.65 M increasingly promoted off-pathway formation of soluble aggregates and at 0.5-0.2 M progressively promoted precipitation, maximal recovery of biologically active protein required a twofold transition in the surrounding guanidine/HCl concentration (6 M-->0.5 M-->0 M). A single additional size-exclusion chromatographic step yielded a final product that was > 99.5% pure, had specific antiviral activity > 10(7) U/mg protein and contained < or = 25 pg/ml endotoxin. Cross-linking by means of disulfosuccinimidyl tartarate revealed that the refolded protein possessed a dimeric structure. Furthermore, we have characterized three different molecular species of recombinant porcine interferon-gamma that are formed under non-optimal refolding conditions (1 M guanidine/HCl) and that differ from each other in specific activity, size and stability. One of these converts irreversibly into dimeric interferon-gamma in a temperature-dependent manner and is therefore considered as a productive folding intermediate.
Vandenbroeck
K
K
Rega Institute, University of Leuven, Belgium.
Martens
E
E
D'Andrea
S
S
Billiau
A
A
D000818:Animals; D015153:Blotting, Western; D002458:Cell Fractionation; D002499:Centrifugation, Density Gradient; D003432:Cross-Linking Reagents; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000378:metabolism; D019791:Guanidine; D006146:Guanidines; D007371:Interferon-gamma / Q000737:chemistry / Q000302:isolation & purification*; D046911:Macromolecular Substances; D017510:Protein Folding; D011994:Recombinant Proteins; D013552:Swine
D003432:Cross-Linking Reagents; D006146:Guanidines; D046911:Macromolecular Substances; D011994:Recombinant Proteins; D007371:Interferon-gamma; D019791:Guanidine