Cysteinylated protein as reactive disulfide: An alternative route to affinity labeling

Zheng Miao, Mark R. McCoy, Diment D. Singh, Brianda Barrios, Oliver L. Hsu, Sarah M. Cheal, Claude F. Meares

Research output: Contribution to journalArticle

5 Citations (Scopus)

Abstract

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.

Original languageEnglish (US)
Pages (from-to)15-19
Number of pages5
JournalBioconjugate Chemistry
Volume19
Issue number1
DOIs
StatePublished - Jan 2008

Fingerprint

Disulfides
Labeling
Ligands
Proteins
Cysteine
Antibodies
Nucleophiles
Monomers
Sulfhydryl Compounds
Bearings (structural)
Artificial Receptors
Antibody Binding Sites
Sulfhydryl Reagents
Protein Array Analysis
Immunoglobulin Fragments
Immunoglobulin Fab Fragments
Binding sites
Macromolecules
Medicine
Pharmaceutical Preparations

ASJC Scopus subject areas

  • Chemistry(all)
  • Organic Chemistry
  • Clinical Biochemistry
  • Biochemistry, Genetics and Molecular Biology(all)
  • Biochemistry

Cite this

Miao, Z., McCoy, M. R., Singh, D. D., Barrios, B., Hsu, O. L., Cheal, S. M., & Meares, C. F. (2008). Cysteinylated protein as reactive disulfide: An alternative route to affinity labeling. Bioconjugate Chemistry, 19(1), 15-19. https://doi.org/10.1021/bc700330j

Cysteinylated protein as reactive disulfide : An alternative route to affinity labeling. / Miao, Zheng; McCoy, Mark R.; Singh, Diment D.; Barrios, Brianda; Hsu, Oliver L.; Cheal, Sarah M.; Meares, Claude F.

In: Bioconjugate Chemistry, Vol. 19, No. 1, 01.2008, p. 15-19.

Research output: Contribution to journalArticle

Miao, Z, McCoy, MR, Singh, DD, Barrios, B, Hsu, OL, Cheal, SM & Meares, CF 2008, 'Cysteinylated protein as reactive disulfide: An alternative route to affinity labeling', Bioconjugate Chemistry, vol. 19, no. 1, pp. 15-19. https://doi.org/10.1021/bc700330j
Miao, Zheng ; McCoy, Mark R. ; Singh, Diment D. ; Barrios, Brianda ; Hsu, Oliver L. ; Cheal, Sarah M. ; Meares, Claude F. / Cysteinylated protein as reactive disulfide : An alternative route to affinity labeling. In: Bioconjugate Chemistry. 2008 ; Vol. 19, No. 1. pp. 15-19.
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