High-Yield Covalent Attachment of Epidermal Growth Factor to Its Receptor by Kinetically Controlled, Stepwise Affinity Cross-Linking

Randall L. Woltjer, Ludmila Weclas-Henderson, James V. Staros, Ioannis A. Papayannopoulos

Research output: Contribution to journalArticle

9 Scopus citations

Abstract

We report here the use of a stepwise affinity cross-linking technique in the specific covalent attachment of epidermal growth factor (EGF) to its receptor. A heterobifunctional cross-linking reagent, sulfo-TV-succinimidyl 4-(fluorosulfonyl)benzoate (SSFSB), which contains a rapidly reacting sulfo-N-succinimidyl active ester and a much more slowly reacting aromatic fluorosulfonyl moiety, was synthesized and characterized. Murine EGF (mEGF) was modified by the cross-linker to yield as the major product a derivative of mEGF having the (fluorosulfonyl)benzoyl moiety attached covalently at the amino terminus. SSFSB-modified, 125I-labeled mEGF was separated from unreacted SSFSB by size-exclusion chromatography and applied to shed membrane vesicles from A431 human carcinoma cells. Binding of derivatized 125I-mEGF to vesicles led to high yields (>60%) of covalent linkage of 125I-mEGF to the EGF receptor, as determined by measurement of the fraction of specifically bound radiolabel which comigrated with the EGF receptor in NaDodS04-polyacrylamide gels. The specificity of affinity cross-linking was evident in the negligible degree of labeling of species other than the EGF receptor and in the retention of EGF-stimulated receptor kinase activity after cross-linking.

Original languageEnglish (US)
Pages (from-to)7341-7346
Number of pages6
JournalBiochemistry
Volume31
Issue number32
DOIs
StatePublished - Feb 1 1992

ASJC Scopus subject areas

  • Biochemistry

Fingerprint Dive into the research topics of 'High-Yield Covalent Attachment of Epidermal Growth Factor to Its Receptor by Kinetically Controlled, Stepwise Affinity Cross-Linking'. Together they form a unique fingerprint.

  • Cite this