Cairns M T, Green A J, White P M, Johnston P G, Brenner S
Department of Medicine, University of Cambridge, UK.
Gene. 1997 Jan 31;185(1):5-9. doi: 10.1016/s0378-1119(96)00601-4.
A bacterial expression vector is described for investigation of protein-protein interactions. Important features of the vector include partition of the cI repressor of bacteriophage lambda into two functional domains separated by a multicloning site, and low level auto-regulated expression of human genes as C-terminal fusions to the DNA-binding domain of cI. Two different reporter systems have been employed; expression of either a suppressor tRNA or the alkaline phosphatase gene is dependent in both cases on the extent of repression of the major leftward promoter of lambda (lambdaP(L)). The cAMP-dependent protein kinase (PKA) has been used as a model protein complex because both homodimer and heterodimer interactions are known to occur and because cAMP acts as a modulator of these interactions. It has been shown that the product of the repressor gene with newly incorporated expressed polylinker restriction sites still functions as a repressor. Substitution of the dimerisation domain of the cI repressor with the regulatory subunit of PKA does not diminish the ability of a cI fusion protein to repress expression of the reporter gene from lambdaP(L), indicating that the regulatory subunit of PKA dimerises the fusion protein in the Escherichia coli cytoplasm. Substitution instead with the catalytic subunit of PKA destroys the repression ability of cI, which is partially restored by separate expression of the regulatory subunit within the same cell. Complete restoration is achieved using a host E. coli strain which has lost its ability to synthesise cAMP and again this can be reversed by the addition of exogenous cAMP to these cells. Human PKA has been reconstituted in the E. coli cytoplasm, where all subunit interactions appear functional and respond as expected to the allosteric modulator cAMP.
描述了一种用于研究蛋白质 - 蛋白质相互作用的细菌表达载体。该载体的重要特征包括将噬菌体λ的cI阻遏蛋白分为由多克隆位点隔开的两个功能域，以及人类基因作为cI DNA结合域的C端融合蛋白的低水平自动调节表达。采用了两种不同的报告系统；在这两种情况下，抑制性tRNA或碱性磷酸酶基因的表达均取决于λ主要左向启动子（λP(L)）的抑制程度。依赖于cAMP的蛋白激酶（PKA）已被用作模型蛋白复合物，因为已知会发生同二聚体和异二聚体相互作用，并且因为cAMP作为这些相互作用的调节剂。已经表明，带有新掺入的表达多克隆位点限制酶切位点的阻遏基因产物仍作为阻遏蛋白发挥作用。用PKA的调节亚基替代cI阻遏蛋白的二聚化结构域不会削弱cI融合蛋白抑制来自λP(L)的报告基因表达的能力，这表明PKA的调节亚基在大肠杆菌细胞质中使融合蛋白二聚化。相反，用PKA的催化亚基替代会破坏cI的抑制能力，通过在同一细胞中单独表达调节亚基可部分恢复该能力。使用已丧失合成cAMP能力的宿主大肠杆菌菌株可实现完全恢复，并且通过向这些细胞中添加外源性cAMP可再次逆转这种情况。人类PKA已在大肠杆菌细胞质中重建，其中所有亚基相互作用似乎都起作用，并如预期那样对变构调节剂cAMP作出反应。
