Functional aspects of co-variant surface charges in an antibody fragment.
A mutational analysis of three co-variant pairs of residues, located at the surface of a single-chain fragment, variable (scFv), remote from the antigen-binding site, was performed to investigate the tolerance of these positions to amino acid changes. The replacements consisted of the elimination or addition of charges, or in their replacement by a charge of opposite sign. As measured by Biacore, antigen-binding kinetics and specificity were essentially unaffected by the mutations. The purified scFvs remained mostly 100% active for 14 h, and their sensitivity to guanidinium-chloride denaturation was similar. These observations indicate that the mutations did not affect antigen-binding properties and that protein folding was conserved. However, the various scFvs differed greatly in half-life in periplasmic extracts (<4 h to >16 h at 25 degrees C). The deleterious effect on half-life produced by single mutations could be reversed by introducing a second mutation that restores the natural combination of amino acids in the co-variant pair, indicating that the consequence of charge modifications at these locations depends on the sequence context. We propose that the differences in half-life result from differences in aggregation propensities with other periplasmic proteins, related to the presence of charged patches at the surface of the scFvs. The practical implication is that changes in surface charge may drastically affect the level of active molecules in complex protein mixtures, a potentially important consideration in engineering scFvs for biotechnological or medical purposes.
Hugo
Nicolas
N
Biotechnologie des Interactions Moléculaires Ecole Supérieure de Biotechnologie de Strasbourg, Pôle API, Bld Sébastien Brant, 67400 Illkirch, France.
Lafont
Virginie
V
Beukes
Mervyn
M
Altschuh
Danièle
D
D000937:Antigen-Antibody Reactions; D000941:Antigens / Q000276:immunology; D019791:Guanidine; D006207:Half-Life; D007135:Immunoglobulin Variable Region / Q000737:chemistry* / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010455:Peptides / Q000276:immunology; D011485:Protein Binding; D011489:Protein Denaturation; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013499:Surface Properties
D000941:Antigens; D007135:Immunoglobulin Variable Region; D010455:Peptides; D011994:Recombinant Proteins; D019791:Guanidine