Maier Christina J, Maier Richard H, Hintner Helmut, Bauer Johann W, Onder Kamil
Department of Cell Biology, University of Salzburg/Naturwissenschaftliche Fakultät, Salzburg, Austria.
Assay Drug Dev Technol. 2010 Oct;8(5):625-9. doi: 10.1089/adt.2009.0266. Epub 2010 Jun 1.
The yeast 2-hybrid (Y2H) system is a powerful method for identifying protein-protein interactions (PPIs), requiring minimal prior information of the putative interactors. Currently available automated versions of the Y2H system are sufficiently developed to allow facile genome-wide PPI screening to compile extensive inter-actome data. A limitation of the Y2H approach, however, is that all primary hits have to be technically verified and biologically evaluated by complementary methods, which is time-consuming, costly, and laborious. Furthermore, the yeast intracellular environment can lead to spurious results for proteins of other organisms, for example because of differences in post-translational modifications or the presence/absence of bridging proteins. Many researchers now confirm PPIs found in the Y2H system by retesting the candidates in the mammalian 2-hybrid system (M2H). However, although such combined Y2H-M2H testing is desirable and perhaps necessary, recloning of Y2H candidates into M2H vectors, especially on a large scale, is time-consuming and costly. To address this shortcoming, we introduce here a new site-specific recombination-capable M2H vector system that is fully compatible with the site-specific Y2H system that we recently described in Biotechniques [2008;45(3):235-244]. The results show that the new vectors are: (a) Gateway®, compatible and suitable for fast cloning; (b) fully functional in the M2H system without influencing the capacity of the selection system or creating autoactivators; and (c) directly compatible with the existing site-specific Y2H vector system, as demonstrated by confirmation of Y2H PPI candidates in the M2H system.
酵母双杂交（Y2H）系统是一种用于鉴定蛋白质-蛋白质相互作用（PPI）的强大方法，对假定的相互作用蛋白所需的先验信息极少。目前可用的Y2H系统自动化版本已得到充分发展，能够轻松进行全基因组PPI筛选，以汇编广泛的相互作用组数据。然而，Y2H方法的一个局限性在于，所有初步筛选出的相互作用蛋白都必须通过互补方法进行技术验证和生物学评估，这既耗时、成本高又费力。此外，酵母细胞内环境可能会导致其他生物体蛋白质产生假阳性结果，例如由于翻译后修饰的差异或桥接蛋白的存在与否。现在许多研究人员通过在哺乳动物双杂交系统（M2H）中重新测试候选蛋白来确认在Y2H系统中发现的PPI。然而，尽管这种Y2H-M2H联合测试是可取的，甚至可能是必要的，但将Y2H候选蛋白重新克隆到M2H载体中，尤其是大规模克隆，既耗时又成本高。为了解决这一缺点，我们在此引入一种新的具有位点特异性重组能力的M2H载体系统，该系统与我们最近在《生物技术》[2008；45（3）：235 - 244]中描述的位点特异性Y2H系统完全兼容。结果表明，新载体具有以下特点：（a）与Gateway®兼容，适合快速克隆；（b）在M2H系统中功能完全正常，不影响选择系统的能力，也不会产生自激活因子；（c）与现有的位点特异性Y2H载体系统直接兼容，这一点已通过在M2H系统中对Y2H PPI候选蛋白的确认得到证明。
