title: Selective domain stabilization as a strategy to reduce fusion protein aggregation.
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.
authors.affiliation: Department of Chemical and Biological Engineering, University of Colorado Boulder, Boulder, Colorado 80309, USA.
authors.forename: Amanda A
authors.initials: AA
authors.lastname: Cordes
authors.forename: Christopher W
authors.initials: CW
authors.lastname: Platt
authors.forename: John F
authors.initials: JF
authors.lastname: Carpenter
authors.forename: Theodore W
authors.initials: TW
authors.lastname: Randolph
mesh_terms: D019382:Human Growth Hormone / Q000737:chemistry*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008027:Light; D011487:Protein Conformation; D017510:Protein Folding; D055550:Protein Stability; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000737:chemistry*; D012542:Scattering, Radiation; D012709:Serum Albumin / Q000737:chemistry*
chemical_list: D011993:Recombinant Fusion Proteins; D012709:Serum Albumin; D019382:Human Growth Hormone