Estojak J, Brent R, Golemis E A
Fox Chase Cancer Center, Philadelphia, Pennsylvania 19111, USA.
Mol Cell Biol. 1995 Oct;15(10):5820-9. doi: 10.1128/MCB.15.10.5820.
Since their introduction, the interaction trap and other two-hybrid systems have been used to study protein-protein interactions. Despite their general use, little is known about the extent to which the degree of protein interaction determined by two-hybrid approaches parallels the degree of interaction determined by biochemical techniques. In this study, we used a set of lexAop-LEU2 and lexAop-lacZ reporters to calibrate the interaction trap. For the calibration, we used two sets of proteins, the Myc-Max-Mxi1 helix-loop-helix proteins, and wild-type and dimerization-defective versions of the lambda cI repressor. Our results indicate that the strength of interaction as predicted by the two-hybrid approach generally correlates with that determined in vitro, permitting discrimination of high-, intermediate-, and low-affinity interactions, but there was no single reporter for which the amount of gene expression linearly reflected affinity measured in vitro. However, some reporters showed thresholds and only responded to stronger interactions. Finally, some interactions were subject to directionality, and their apparent strength depended on the reporter used. Taken together, our results provide a cautionary framework for interpreting affinities from two-hybrid experiments.
自引入以来，相互作用陷阱及其他双杂交系统已被用于研究蛋白质-蛋白质相互作用。尽管它们被广泛使用，但对于双杂交方法所确定的蛋白质相互作用程度与生化技术所确定的相互作用程度的平行程度，人们了解甚少。在本研究中，我们使用了一组lexAop-LEU2和lexAop-lacZ报告基因来校准相互作用陷阱。为了进行校准，我们使用了两组蛋白质，即Myc-Max-Mxi1螺旋-环-螺旋蛋白，以及λ cI阻遏物的野生型和二聚化缺陷型。我们的结果表明，双杂交方法预测的相互作用强度通常与体外测定的结果相关，能够区分高、中、低亲和力相互作用，但没有一个单一的报告基因，其基因表达量能线性反映体外测量的亲和力。然而，一些报告基因显示出阈值，仅对更强的相互作用有反应。最后，一些相互作用存在方向性，其表观强度取决于所使用的报告基因。综合来看，我们的结果为解释双杂交实验中的亲和力提供了一个警示框架。
