C-terminal labeling of immunoglobulin G with a cysteine derivative by carboxypeptidase Y catalyzed transpeptidation

We describe a new method for the site-specific incorporation of an extrinsi c cysteine to the C-termini of immunoglobulin G (IgG) using carboxypeptidas e Y (CPase Y) catalyzed transpeptidation. The transpeptidase activity of CP ase Y was employed to attach cysteine esters to the C-termini of the IgG mo lecule (cysteinylation) at alkaline pH. No CPase Y catalyzed transpeptidati on products were found when native IgG was used as the substrate or when cy steine was used as the nucleophile. However, C-terminal labeling occurred w hen cysteine ethyl eater (CysOEt) or cysteine isobutyl ester (CysOiBu) was used as the nucleophile and IgG methyl ester as the substrate. When CysOiBu was used as the nucleophile, the maximal labeling yield obtained with IgG methyl ester as substrate was 25%, assuming all four C-termini in the IgG m olecule were labeled equally. The C-terminal labeling pattern of cysteinyla ted IgG was determined by autoradiography followed by the integration of ra dio-density. It revealed that both the C-termini of the heavy and light cha ins of IgG methyl ester were labeled with CysOiBu. (C) 2000 Academic Press.