Affinity resins containing enzymatically resistant mRNA cap analogs - A new tool for the analysis of cap-binding proteins

Abstract

Cap-binding proteins have been routinely isolated using m 7GTP-Sepharose; however, this resin is inefficient for proteins such as DcpS (scavenger decapping enzyme), which interacts not only with the 7-methylguanosine, but also with the second cap base. In addition, DcpS purification may be hindered by the reduced resin capacity due to the ability of DcpS to hydrolyze m 7GTP. Here, we report the synthesis of new affinity resins, m 7GpCH 2pp- and m 7GpCH 2ppA-Sepharoses, with attached cap analogs resistant to hydrolysis by DcpS. Biochemical tests showed that these matrices, as well as a hydrolyzable m 7GpppA-Sepharose, bind recombinant mouse eIF4E (28-217) specifically and at high capacity. In addition, purification of cap-binding proteins from yeast extracts confirmed the presence of all expected cap-binding proteins, including DcpS in the case of m 7GpCH 2pp- and m 7GpCH 2ppA-Sepharoses. In contrast, binding studies in vitro demonstrated that recombinant human DcpS efficiently bound only m 7GpCH 2ppA-Sepharose. Our data prove the applicability of these novel resins, especially m 7GpCH 2ppA-Sepharose, in biochemical studies such as the isolation and identification of cap-binding proteins from different organisms.