Kimball S R, Horetsky R L, Jagus R, Jefferson L S
Department of Cellular and Molecular Physiology, The Pennsylvania State University College of Medicine, Hershey, Pennsylvania 17033, USA.
Protein Expr Purif. 1998 Apr;12(3):415-9. doi: 10.1006/prep.1998.0863.
The alpha-subunit of eukaryotic initiation factor eIF2 (eIF2alpha) plays an important role in the regulation of mRNA translation through modulation of the interaction of eIF2 and a second initiation factor, eIF2B. The interaction of the two proteins is regulated in vivo by phosphorylation of eIF2alpha at Ser51. In the present study, rat eIF2alpha was expressed in Sf21 cells using the baculovirus expression system. The recombinant protein was purified to >90% homogeneity in a single immunoaffinity chromatographic step. The protein was free of endogenous eIF2alpha kinase activity and was rapidly phosphorylated by the eIF2alpha kinases HCR and PKR. A variant of eIF2alpha in which the phosphorylation site was changed to Ala was also expressed and purified. The variant eIF2alpha was not phosphorylated by either HCR or PKR, demonstrating that the kinases specifically phosphorylate the correct site in the recombinant protein even in the absence of the other two subunits of the protein. In summary, a rapid and inexpensive method for obtaining eIF2alpha has been developed. Use of the wildtype and variant forms of eIF2alpha to measure eIF2alpha kinase activity in cell and tissue extracts should greatly facilitate examination of the regulation of mRNA translation under a variety of conditions.
真核起始因子eIF2的α亚基（eIF2α）通过调节eIF2与另一个起始因子eIF2B的相互作用，在mRNA翻译调控中发挥重要作用。这两种蛋白质的相互作用在体内受eIF2α第51位丝氨酸磷酸化的调节。在本研究中，使用杆状病毒表达系统在Sf21细胞中表达大鼠eIF2α。重组蛋白在单步免疫亲和色谱中纯化至>90%的纯度。该蛋白无内源性eIF2α激酶活性，并被eIF2α激酶HCR和PKR快速磷酸化。还表达并纯化了一种将磷酸化位点变为丙氨酸的eIF2α变体。该变体eIF2α不被HCR或PKR磷酸化，表明即使在不存在该蛋白的其他两个亚基的情况下，激酶也能特异性地磷酸化重组蛋白中的正确位点。总之，已开发出一种快速且廉价的获取eIF2α的方法。使用野生型和变体形式的eIF2α来测量细胞和组织提取物中的eIF2α激酶活性，应能极大地促进在各种条件下对mRNA翻译调控的研究。
