Nishi N, Morino S, Tomoo K, Youtani T, Ishida T
Department of Physical Chemistry, Osaka University of Pharmaceutical Sciences, Takatsuki.
J Biochem. 1998 Jan;123(1):157-61. doi: 10.1093/oxfordjournals.jbchem.a021903.
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.
编码人起始因子（eIF）4E结合蛋白1（4E-BP1）的人工基因经化学合成和克隆。虽然单独的4E-BP1基因的表达尚未实现，但该基因在大肠杆菌[BL21(DE3)]中作为与谷胱甘肽-S-转移酶（GST）基因的融合基因进行表达，使用的是原核基因融合载体（pGEX-4T-2），其包含编码特定蛋白酶α-凝血酶切割位点的基因序列。融合基因产物通过谷胱甘肽琼脂糖4B亲和柱层析纯化至同质。通过m7GTP和谷胱甘肽亲和层析表明，4E-BP1与eIF-4E的结合能力与它与eIF-4E x m7GTP复合物的结合能力几乎相同，这意味着eIF-4E有不同的结合位点，并且其对4E-BP1和mRNA帽结构的结合是非别构性的。与eIF-4E与mRNA帽结构的结合不同，在后者的结合中一些功能性氨基酸起重要作用，而与4E-BP1的结合被认为是通过多种非特异性相互作用发生的。
