Thermodynamically stable aggregation-resistant antibody domains through directed evolution.
Protein aggregates are usually formed by interactions between unfolded or partially unfolded species, and often occur when a protein is denatured by, for example, heat or low pH. In earlier work, we used a Darwinian selection strategy to create human antibody variable domains that resisted heat aggregation. The repertoires of domains were displayed on filamentous phage and denatured (at 80 degrees C in pH 7.4), and folded domains were selected by binding to a generic ligand after cooling. This process appeared to select for domains with denatured states that resisted aggregation, but the domains only had low free energies of folding (Delta G(N-D)(o)=15-20 kJ/mol at 25 degrees C in pH 7.4). Here, using the same phage repertoire, we have extended the method to the selection of domains resistant to acid aggregation. In this case, however, the thermodynamic stabilities of selected domains were higher than those selected by thermal denaturation (under both neutral and acidic conditions; Delta G(N-D)(o)=26-47 kJ/mol at 25 degrees C in pH 7.4, or Delta G(N-D)(o)=27-34 kJ/mol in pH 3.2). Furthermore, we identified a key determinant (Arg28) that increased the aggregation resistance of the denatured states of the domains at low pH without compromising their thermodynamic stabilities. Thus, the selection process yielded domains that combined thermodynamic stability and aggregation-resistant unfolded states. We suggest that changes to these properties are controlled by the extent to which the folding equilibrium is displaced during the process of selection.
Famm
Kristoffer
K
Centre for Protein Engineering, Medical Research Council Centre, Hills Road, Cambridge CB2 2QH, UK.
Hansen
Lars
L
Christ
Daniel
D
Winter
Greg
G
D000906:Antibodies / Q000737:chemistry*; D001120:Arginine; D001435:Bacteriophages; D019020:Directed Molecular Evolution; D006801:Humans; D007135:Immunoglobulin Variable Region / Q000737:chemistry; D009391:Nephelometry and Turbidimetry; D011489:Protein Denaturation; D020836:Protein Structure, Quaternary; D017434:Protein Structure, Tertiary; D012995:Solubility; D012996:Solutions; D013816:Thermodynamics
D000906:Antibodies; D007135:Immunoglobulin Variable Region; D012996:Solutions; D001120:Arginine