Guo Lian-Wang, Assadi-Porter Fariba M, Grant Jennifer E, Wu Hai, Markley John L, Ruoho Arnold E
Department of Pharmacology, University of Wisconsin, Madison, WI 53706, USA.
Protein Expr Purif. 2007 Feb;51(2):187-97. doi: 10.1016/j.pep.2006.07.012. Epub 2006 Jul 22.
Interactions between the transducin alpha-subunit (Galpha(t)) and the cGMP phosphodiesterase gamma-subunit (PDEgamma) are critical not only for turn-on but also turn-off of vertebrate visual signal transduction. Elucidation of the signaling mechanisms dominated by these interactions has been restrained by the lack of atomic structures for full-length Galpha(t)/PDEgamma complexes, in particular, the signaling-state complex represented by Galpha(t).GTPgammaS/PDEgamma. As a preliminary step in our effort for NMR structural analysis of Galpha(t)/PDEgamma interactions, we have developed efficient protocols for the large-scale production of recombinant Galpha(t) (rGalpha(t)) and homogeneous and functional isotopically labeled PDEgamma from Escherichia coli cells. One-step purification of rGalpha(t) was achieved through cobalt affinity chromatography in the presence of glycerol, which effectively removed the molecular chaperone DnaK that otherwise persistently co-purified with rGalpha(t). The purified rGalpha(t) was found to be functional in GTPgammaS/GDP exchange upon activation of rhodopsin and was used to form a signaling-state complex with labeled PDEgamma, rGalpha(t). GTPgammaS/[U-13C,15N]PDEgamma. The labeled PDEgamma sample yielded a well-resolved 1H-15N HSQC spectrum. The methods described here for large-scale production of homogeneous and functional rGalpha(t) and isotope-labeled PDEgamma should support further NMR structural analysis of the rGalpha(t)/PDEgamma complexes. In addition, our protocol for removing the co-purifying DnaK contaminant may be of general utility in purifying E. coli-expressed recombinant proteins.
转导素α亚基（Galpha(t)）与cGMP磷酸二酯酶γ亚基（PDEgamma）之间的相互作用不仅对脊椎动物视觉信号转导的开启至关重要，对其关闭也同样关键。由于缺乏全长Galpha(t)/PDEgamma复合物的原子结构，特别是由Galpha(t).GTPgammaS/PDEgamma代表的信号转导状态复合物，这些相互作用所主导的信号转导机制的阐明受到了限制。作为我们对Galpha(t)/PDEgamma相互作用进行核磁共振结构分析工作的初步步骤，我们开发了从大肠杆菌细胞大规模生产重组Galpha(t)（rGalpha(t)）以及均一且具有功能的同位素标记PDEgamma的有效方案。通过在甘油存在下的钴亲和层析实现了rGalpha(t)的一步纯化，这有效地去除了分子伴侣DnaK，否则它会一直与rGalpha(t)共纯化。发现纯化后的rGalpha(t)在视紫红质激活后进行GTPgammaS/GDP交换时具有功能，并用于与标记的PDEgamma形成信号转导状态复合物，即rGalpha(t).GTPgammaS/[U-13C,15N]PDEgamma。标记的PDEgamma样品产生了分辨率良好的1H-15N HSQC谱。这里描述的大规模生产均一且具有功能的rGalpha(t)和同位素标记PDEgamma的方法应有助于对rGalpha(t)/PDEgamma复合物进行进一步的核磁共振结构分析。此外，我们去除共纯化的DnaK污染物的方案在纯化大肠杆菌表达的重组蛋白方面可能具有普遍用途。
