ABSTRACT
A human serum albumin-human growth hormone (HSA-hGH) fusion protein was used as a model to understand the contributions of individual domains to the aggregation behavior of the overall fusion protein. Aggregation of HSA-hGH was studied at two different pH conditions, pH 5 and pH 7. Conformational stability of the HSA domain was modulated by addition of octanoic acid, a binding ligand. Conformational stability of the fusion protein and the HSA domain were determined from experimentally measured values for free energies of unfolding (ΔG(unf) ) with midpoint of apparent unfolding temperatures (T(m) ) used as surrogate in some cases. Apparent T(m) s of both HSA and HSA-hGH were increased by octanoic acid binding. Osmotic second virial coefficients were measured to monitor protein-protein interactions in solution. Reductions in rates of aggregation were observed under solution conditions that increased protein-protein repulsive interactions even when no changes in conformational stability were detected. The results indicate that colloidal instabilities are responsible for HSA-hGH aggregation and that conformational stability of the HSA domain does not play a dominant role in the aggregation of HSA-hGH.
