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Quantification of human dopamine D2s receptor interactions with G alpha(i,1,2)- and G alpha(o)-proteins.

机译:定量人类多巴胺D2s受体与G alpha(i,1,2)-和G alpha(o)蛋白的相互作用。

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摘要

A simple and rapid in vitro method for qualitative and quantitative estimation of the G alpha-subunits interaction with the third intracellular loop of human D2s dopamine receptor has been developed. For this purpose, D2s-CL3 was cloned in pGEX-2T vector and expressed in E. coli BL21 DE3 as a fusion protein with glutathione-S-transferase (D2s-CL3-GST). The resulting soluble construct was purified by affinity chromatography on glutathione-Sepharose. G alpha-subunits were expressed and purified as His-tagged proteins. For the assay of G alpha/D2s-CL3-GST interactions, varying concentrations of pure His-tagged G alpha-proteins were immobilized on His-Bind Resin and titrated with D2s-CL3-GST fusion protein. G alpha/D2s-CL3-GST interactions were quantified by GST activity determination assay. It was shown that the fusion protein interacts specifically with different G alpha proteins, especially with G alpha(i) proteins. Based on saturation binding analyses, Kd values were determined revealing the highest affinity of His-G alpha(i,2) binding to the fusion protein. The affinities for G alpha(i)/D2s-CL3-GST protein interactions estimated in this way were in nanomolar range of concentrations.
机译:已经开发了一种简单,快速的体外方法,用于定性和定量估计G alpha亚基与人D2s多巴胺受体第三胞内环的相互作用。为此,将D2s-CL3克隆到pGEX-2T载体中,并在大肠杆菌BL21 DE3中表达为与谷胱甘肽-S-转移酶(D2s-CL3-GST)的融合蛋白。通过在谷胱甘肽-Sepharose上的亲和层析纯化所得的可溶性构建体。 G alpha亚基被表达并纯化为His标记蛋白。为了测定G alpha / D2s-CL3-GST相互作用,将不同浓度的纯His-标记的G alpha蛋白固定在His-Bind树脂上,并用D2s-CL3-GST融合蛋白滴定。通过GST活性测定法对G alpha / D2s-CL3-GST相互作用进行了定量。结果表明,融合蛋白与不同的G alpha蛋白,特别是与G alpha(i)蛋白发生特异性相互作用。基于饱和结合分析,确定Kd值,揭示His-G alpha(i,2)与融合蛋白结合的最高亲和力。以这种方式估计的G alpha(i)/ D2s-CL3-GST蛋白相互作用的亲和力在浓度的纳摩尔范围内。

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