Expression of a synthetic gene for initiation factor 4E-binding protein 1 in Escherichia coli and its interaction with eIF-4E and eIF-4E x m7GTP complex.
An artificial gene coding for the human initiation factor (eIF) 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, alpha-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 x 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.
Nishi
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N
Department of Physical Chemistry, Osaka University of Pharmaceutical Sciences, Takatsuki.
Morino
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S
Tomoo
K
K
Youtani
T
T
Ishida
T
T
D000595:Amino Acid Sequence; D001483:Base Sequence; D002352:Carrier Proteins; D003001:Cloning, Molecular; D004274:DNA, Recombinant; D004926:Escherichia coli; D039561:Eukaryotic Initiation Factor-4E; D015870:Gene Expression; D005813:Genes, Synthetic; D008969:Molecular Sequence Data; D009154:Mutation; D010448:Peptide Initiation Factors / Q000378:metabolism*; D010750:Phosphoproteins / Q000235:genetics* / Q000378:metabolism; D011485:Protein Binding; D012314:RNA Cap Analogs / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism
D002352:Carrier Proteins; D004274:DNA, Recombinant; D039561:Eukaryotic Initiation Factor-4E; D010448:Peptide Initiation Factors; D010750:Phosphoproteins; D012314:RNA Cap Analogs; D011993:Recombinant Fusion Proteins; C017516:7-methylguanosine triphosphate