IMPROVED REFOLDING OF AN IMMOBILIZED FUSION PROTEIN

Fusion proteins of monomeric alpha-glucosidase from Saccharomyces cere visiae containing N- or C-terminal hexa-arginine peptides were express ed in the cytosol of Escherichia coli in soluble form. The polycationi c peptide moieties allow noncovalent binding of the denatured fusion p roteins to a polyanionic solid support. Upon removal of the denaturant , refolding of the matrix-bound protein can proceed without perturbati on by aggregation. However, nonspecific interactions of the denatured polypeptide, or of folding intermediates, with the matrix cause a dras tic decrease in renaturation under suboptimal folding conditions. At l ow salt concentrations, ionic interactions of the refolding polypeptid e with the matrix result in lower yields of renaturation. At higher sa lt concentrations, renaturation is prevented by hydrophobic interactio ns with the matrix. Apart from ionic strength, renaturation of the den atured matrix-bound fusion protein must be optimized with respect to p H, temperature, cosolvents, and matrix material used. Under optimum co nditions, immobilized alpha-glucosidase can be renatured with a high y ield at protein concentrations up to 5 mg/ml, whereas folding of the w ild-type enzyme in solution is feasible only at an extremely low prote in concentration (15 mu g/ml). Thus, folding of the immobilized alpha- glucosidase allows an extremely high yield of the renaturated model pr otein. The technology should be applicable to other proteins that tend to aggregate during refolding.