Rapid identification of reactive cysteine residues for site-specific labeling of antibody-Fabs

摘要: Cysteines with reactive thiol groups are attractive tools for site-specific labeling of proteins. Engineering a cysteine residue into proteins multiple disulfide bonds is often challenging task as it may interfere structural and functional properties the protein. Here we developed phage display-based biochemical assay, PHESELECTOR (Phage ELISA Selection Reactive Thiols) to rapidly screen on antibody fragments without interfering their antigen binding, using trastuzumab-Fab (hu4D5Fab) model system. The solvent accessibility values all amino acid residues in hu4D5Fab were calculated available crystal structure information. Serine, alanine valine highest selected tested compare structure-based design method. Cysteine substitutions at partially solvent-accessible or exhibited better reactivity than serine residues. poor correlation between fractional engineered variants indicated value assay identify antibody-Fab surface. Mass spectrometric analysis biotinylated ThioFab (Fab cysteine) confirmed that conjugation occurred only thiols either light heavy chains. ThioFabs constant domains (CL CH(1)) should allow universal application antibody-Fabs.