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

半胱氨酸 化学 缬氨酸 丝氨酸 丙氨酸 硫醇 氨基酸 生物化学 残留物(化学) 噬菌体展示 脱氢丙氨酸 立体化学 组合化学
作者
Jagath R. Junutula,Sunil Bhakta,Helga Raab,Karen E. Ervin,Charles Eigenbrot,Richard Vandlen,Richard H. Scheller,Henry B. Lowman
出处
期刊:Journal of Immunological Methods [Elsevier]
卷期号:332 (1-2): 41-52 被引量:160
标识
DOI:10.1016/j.jim.2007.12.011
摘要

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