Cysteinylated protein as reactive disulfide: an alternative route to affinity labeling.
Engineering the permanent formation of a receptor-ligand complex has a number of promising applications in chemistry, biology, and medicine. Antibodies and other proteins can be excellent receptors for synthetic ligands such as probes or drugs. Because proteins possess an array of nucleophilic sites, the placement of an electrophile on the synthetic ligand to react with a nucleophile on the macromolecule is a standard practice. Previously, we have used the site-directed incorporation of cysteine nucleophiles at the periphery of an antibody's binding site, paired with the chemical design of weakly electrophilic ligands, to produce receptor-ligand pairs that conjugate specifically and permanently (Corneillie et al. (2004) Bioconjugate Chem. 15, 1392-1402 and references therein). After protein expression in Drosophila S2 cells, we found, as is frequently observed, that the engineered cysteine was reversibly blocked by disulfide linkage to a cysteine monomer (cysteinylated). Removal of the cysteine monomer requires some care because of the need to preserve other disulfide linkages in the protein. Here, we report that cysteinylation can be used to advantage by treating the cysteine monomer as a leaving group and the protein disulfide as an electrophile with special affinity for thiols. Two ligands bearing thiol side chains were synthesized and incubated with the cysteinylated antibody Fab fragment 2D12.5 G54C, with the finding that both ligands become covalently attached within a few minutes under physiological conditions. The attachment is robust even in the presence of excess thiol reagents. This rapid, specific conjugation is particularly interesting for biomedical applications.
Department of Chemistry, University of California, Davis, California 95616, USA.
Zheng
Z
Miao
Mark R
MR
McCoy
Diment D
DD
Singh
Brianda
B
Barrios
Oliver L
OL
Hsu
Sarah M
SM
Cheal
Claude F
CF
Meares
D000345:Affinity Labels / Q000737:chemistry*; D000818:Animals; D001665:Binding Sites; D003545:Cysteine / Q000737:chemistry*; D004220:Disulfides / Q000737:chemistry*; D007140:Immunoglobulin Fab Fragments / Q000737:chemistry; D008024:Ligands; D008958:Models, Molecular; D017434:Protein Structure, Tertiary; D012680:Sensitivity and Specificity; D013438:Sulfhydryl Compounds / Q000737:chemistry
D000345:Affinity Labels; D004220:Disulfides; D007140:Immunoglobulin Fab Fragments; D008024:Ligands; D013438:Sulfhydryl Compounds; D003545:Cysteine