REFOLDING AND OXIDATION OF RECOMBINANT HUMAN STEM-CELL FACTOR PRODUCED IN ESCHERICHIA-COLI

Oxidative folding of recombinant human stem cell factor (rhSCF) produc ed in Escherichia coli was investigated in vitro. Folding of denatured and reduced rhSCF involves at least five intermediate forms, I-1 to I -5, detectable by their differences in hydrophobicity using reverse-ph ase high performance liquid chromatography. Both I-1 and I-2 contain a native-like disulfide bond, Cys(4)-Cys(89) and Cys(43)-Cys(138), resp ectively, and I-3 forms a mispaired disulfide, Cys(43)-Cys89. These fo rms appear to reach steady state equilibrium and are important folding intermediates. I-1 was found to be the prominent intermediate that di rectly folds into native rhSCF (N); and the thermodynamically less sta ble I-2 favors rearrangment into I-1. I-3 may serve as an intermediate for disulfide rearrangement between I-1 and I-2, I-4 and I-5, which a re disulfide-linked dimers, are in equilibrium with reduced rhSCF and other intermediates and may not play an important role in rhSCF foldin g. Both trifluoroacetic acid-trapped I-1 and I-2, after isolation by h igh performance liquid chromatography, proceed with the remaining oxid ative folding process after reconstitution. Iodoacetate-trapped I-1 an d I-2 contain low alpha-helical content and some tertiary structure, w hile I-3 and reduced rhSCF have little ordered structure. Gel filtrati on/light-scattering experiments indicate that reduced rhSCF and iodoac etate-trapped I-1, I-2, and I-3 exist as dimeric forms, indicating tha t rhSCF dimerization precedes formation of disulfide bonds. I-I, I-2, I-3, and the C43,138A analog lacking Cys(43)-Cys(138) bond are not bio logically active or exhibit significantly lower activity. The two disu lfide bonds in rhSCF seem to be essential for the molecule to maintain an active conformation required for its receptor binding and biologic al activities.