Expression of a Synthetic Gene for Initiation Factor 4E-Binding Protein 1 in Escherichia coli and Its Interaction with eIF-4E and eIF-4E-m7GTP Complex

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Abstract
An artificial gene coding for the human initiation factor (elF) 4E-binding protein 1 (4E-BP1) was chemically synthesized and cloned. Although the expression of the 4E-BP1 gene alone has not yet been accomplished, the gene was expressed in Escherichia coli [BL21(DE3)] as a fusion gene with the glutathione-S-transferase (GST) gene using a prokaryotic gene fusion vector (pGEX-4T-2), which contains a gene sequence coding the cleavage site for a specific protease, α-thrombin. The fusion gene product was purified to homogeneity by glutathione Sepharose-4B affinity column chromatography. It was shown by m7GTP- and glutathione-affinity chromatography that the binding ability of 4E-BP1 to eIF-4E is nearly the same as that to the eIF-4E-m7GTP complex, implying different binding sites of eIF-4E and its nonallosteric obligation for 4E-BP1 and mRNA cap structure. In contrast with the binding of eIF-4E to the mRNA cap structure, where some functional amino acids play an important role in the binding, the binding to 4E-BP1 was suggested to occur via multiple nonspecific interactions.

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