Design and Preparation of Affinity Columns for the Purification of Eukaryotic Messenger Ribonucleic Acid Cap Binding Protein

Kathleen M. Rupprecht, Sidney M. Hecht, Nahum Sonenberg, Aaron J. Shatki

Research output: Contribution to journalArticle

9 Scopus citations

Abstract

2’,3'-O-[l-(2-Carboxyethyl)ethylidene]-7-methylguanosine 5'-diphosphate (5) and 7-(5-carboxypentyl) guanosine 5'-diphosphate (13) have been synthesized and immobilized on AH-Sepharose 4B to the extent of 17.4 and 36.6µmol of ligand/g of gel, respectively. The affinity resins thus derived were employed in columns for the purification of 24K cap binding protein (CBP) from rabbit reticulocytes. Each resin was found to retain the protein of interest; elution of 24K CBP could then be effected by washing with 70µM m7GDP. The 24K CBPs released from both columns were found to be active, both as judged by a cross-linking assay that utilized IO4-oxidized methyl-3 3H-labeled reovirus mRNA as a substrate for the protein and also by the ability of the isolated 24K CBP to stimulate the translation of capped Sindbis virus mRNA in HeLa cell extracts.

Original languageEnglish (US)
Pages (from-to)6570-6577
Number of pages8
JournalBiochemistry
Volume20
Issue number23
DOIs
StatePublished - Nov 1981
Externally publishedYes

ASJC Scopus subject areas

  • Biochemistry

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