Design and preparation of affinity columns for the purification of eukaryotic messenger ribonucleic acid cap binding...

2',3'-O-[1-(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 36.6 mumol of ligand/g of gel, respectively. The affinity resins thus derives were employed in columns for the purificaton 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 70 microM m7GDP. The 24K CBPs released from both columns were found to be active, both as judged by a cross-linking assay that utilized 10(4)-oxidized methyl-3H-labeled reovirus mRNA as a substrate for the protein and also by the ability of the isolated 24K CBP to stimulate the translocation of capped Sindbis virus mRNA in HeLa cell extracts.