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 extrinsic cysteine to the C-termini of immunoglobulin G (IgG) using carboxypeptidase Y (CPase Y) catalyzed transpeptidation. The transpeptidase activity of CPase Y was employed to attach cysteine esters to the C-termini of the IgG molecule (cysteinylation) at alkaline pH. No CPase Y catalyzed transpeptidation products were found when native IgG was used as the substrate or when cysteine was used as the nucleophile. However, C-terminal labeling occurred when cysteine ethyl ester (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 molecule were labeled equally. The C-terminal labeling pattern of cysteinylated IgG was determined by autoradiography followed by the integration of radiodensity. It revealed that both the C-termini of the heavy and light chains of IgG methyl ester were labeled with CysOiBu.
Lin
S
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Centre for Optoelectronic Biomedicine, National Taiwan University, Taipei, 100, Taiwan.
Lowe
C R
CR
D002268:Carboxypeptidases / Q000737:chemistry*; D002384:Catalysis; D043402:Cathepsin A; D003545:Cysteine / Q000737:chemistry*; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D007074:Immunoglobulin G / Q000737:chemistry*; D010455:Peptides / Q000737:chemistry*
D007074:Immunoglobulin G; D010455:Peptides; D002268:Carboxypeptidases; D043402:Cathepsin A; D003545:Cysteine