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 col i, was found to accumulate in cytoplasmic inclusion bodies. The influe nce of various physicochemical parameters on refolding was investigate d using 6 M guanidine/HCl-solubilised inclusion bodies which had been purified by ultracentrifugation on a sucrose step gradient. It appeare d that the yield of reconstitution of denatured protein reached 60-70% under optimum conditions, i.e. at an intermediary guanidine/HCl conce ntration of 0.5 M and at a protein concentration of 10-20 muM (0-degre es-C). Since intermediary guanidine/HCl concentrations at 0.5 --> 1.65 M increasingly promoted off-pathway formation of soluble aggregates a nd at 0.5-0.2 M progressively promoted precipitation, maximal recovery of biologically active protein required a twofold transition in the s urrounding guanidine/HCl concentration (6 M --> 0.5 M --> 0 M). A sing le additional size-exclusion chromatographic step yielded a final prod uct that was >99.5% pure, had specific antiviral activity > 10(7) U/mg protein and contained less-than-or-equal-to 25 pg/ml endotoxin. Cross -linking by means of disulfosuccinimidyl tartarate revealed that the r efolded protein possessed a dimeric structure. Furthermore, we have ch aracterized three different molecular species of recombinant porcine i nterferon-y 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 i nterferon-gamma in a temperature-dependent manner and is therefore con sidered as a productive folding intermediate.