Hagedorn C H, Spivak-Kroizman T, Friedland D E, Goss D J, Xie Y
Department of Medicine, Emory University School of Medicine, Atlanta, Georgia 30322, USA.
Protein Expr Purif. 1997 Feb;9(1):53-60. doi: 10.1006/prep.1996.0661.
Protein-mRNA cap interactions represent a critical point for regulating gene expression in vivo. For example, a rapid stimulation of gene expression at the mRNA level is mediated by insulin regulating the availability of functional cap-binding protein (eIF-4E). In addition, several viruses modify cap binding proteins to regulate host vs viral gene expression. However, little is known about the molecular details of eIF-4E interactions with m7GTP mRNA caps, with regulatory proteins (e.g., eIF-4E binding proteins), and with proteins within the eIF-4F complex. To study these protein-mRNA and protein-protein interactions in mammalian systems we have constructed a T7 polymerase-driven expression vector containing the coding sequence for human eIF-4E. Recombinant eIF-4Ehuman was purified in a functional state by m7GTP affinity chromatography and Mono Q FPLC. This recombinant protein has biological and physical characteristics that are similar or identical to native eIF-4E. Fluorescence titration studies determined the equilibrium constant for recombinant eIF-4E/m7GTP binding to be 10.1 +/- 0.3 x 10(5) M(-1). To isolate eIF-4E binding proteins, recombinant eIF-4E was linked to agarose beads and incubated with cell lysates. Several proteins were isolated, including a 220-kDa protein that was confirmed to be the p220 subunit of eIF-4F by its proteolysis during incubation with lysates of poliovirus-infected cells. We conclude that recombinant eIF-4E produced in Escherichia coli provides a useful tool for studying eIF-4E/protein and eIF-4E/mRNA cap interactions and their role in regulating mammalian gene expression.
蛋白质与mRNA帽的相互作用是体内调节基因表达的关键点。例如，胰岛素通过调节功能性帽结合蛋白（eIF-4E）的可用性，在mRNA水平上快速刺激基因表达。此外，几种病毒会修饰帽结合蛋白以调节宿主与病毒基因的表达。然而，关于eIF-4E与m7GTP mRNA帽、调节蛋白（如eIF-4E结合蛋白）以及eIF-4F复合物中的蛋白质之间相互作用的分子细节，我们所知甚少。为了研究哺乳动物系统中的这些蛋白质与mRNA以及蛋白质与蛋白质之间的相互作用，我们构建了一个由T7聚合酶驱动的表达载体，其中包含人类eIF-4E的编码序列。通过m7GTP亲和层析和Mono Q快速蛋白质液相色谱法，以功能状态纯化了重组人eIF-4E。这种重组蛋白具有与天然eIF-4E相似或相同的生物学和物理特性。荧光滴定研究确定重组eIF-4E/m7GTP结合的平衡常数为10.1±0.3×10⁵ M⁻¹。为了分离eIF-4E结合蛋白，将重组eIF-4E与琼脂糖珠连接，并与细胞裂解物孵育。分离出了几种蛋白质，其中一种220 kDa的蛋白质，在与脊髓灰质炎病毒感染细胞的裂解物孵育期间通过蛋白水解被确认为eIF-4F的p220亚基。我们得出结论，在大肠杆菌中产生的重组eIF-4E为研究eIF-4E/蛋白质和eIF-4E/mRNA帽相互作用及其在调节哺乳动物基因表达中的作用提供了一个有用的工具。
