AGGREGATION PATHWAY OF RECOMBINANT HUMAN KERATINOCYTE GROWTH-FACTOR AND ITS STABILIZATION

Recombinant human keratinocyte growth factor (rhKGF) is prone to aggre gation at elevated temperatures. Its aggregation pathway is proposed t o proceed initially with a conformational change which perhaps results from repulsion between positively charged residues in clusters formin g heparin binding sites. Unfolding of the protein leads to formation o f large soluble aggregates. These soluble aggregates then form disulfi de cross-linked precipitates. Finally these precipitates are converted to scrambled disulfides and/or non-disulfide cross-linked precipitate s. Stabilizers such as heparin, sulfated polysaccharides, anionic poly mers and citrate can greatly decrease the rate of aggregation of rhKGF at elevated temperatures. These molecules may all act by reducing cha rge repulsion on the protein thus stabilizing the native conformation. EDTA, on the other hand, is found to inhibit disulfide formation in a ggregates and has only a moderate stabilizing effect on rhKGF.