An evaluation of in vitro protein-protein interaction techniques:: Assessing contaminating background proteins
An evaluation of in vitro protein-protein interaction techniques:: Assessing contaminating background proteins
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DOI：
10.1002/pmic.200500517
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发表时间：
2006-04-01
期刊：
影响因子：
3.4
通讯作者：
Turner, RJ
中科院分区：
文献类型：
--
作者：
Howell, JM;Winstone, TL;Turner, RJ
蛋白质-蛋白质相互作用的确定是在更广泛的细胞背景下分配功能和辨别蛋白质的生物学相关性的重要组成部分。体外蛋白质-蛋白质相互作用的方法，包括亲和层析，共沉淀，和较新的方法，如蛋白质芯片阵列，在蛋白质相互作用的检测，特别是在良好表征的生物体与测序的基因组中，有很大的希望。然而，这些方法中的每一种都会吸引某些背景蛋白，这些背景蛋白会阻碍对真正相互作用物的检测和鉴定。此外，大肠杆菌中表达的重组蛋白也被广泛用于评估蛋白质-蛋白质相互作用，因此这些分离株中的背景蛋白可能污染相互作用研究。因此，对真实相互作用的严格验证不仅需要通过替代技术发现相互作用，而且更重要的是，研究人员要意识到并控制基质/支持依赖性。在这里，我们评估这些方法与DmsD（一个E。coli氧化还原酶成熟蛋白伴侣），体外培养，采用E. coli亚细胞组分作为猎物来源。我们比较和对比各种体外相互作用方法，以确定一些背景蛋白和蛋白质谱，这是固有的每种方法在E。coli系统。
Determination of protein-protein interactions is an important component in assigning function and discerning the biological relevance of proteins within a broader cellular context. In vitro protein-protein interaction methodologies, including affinity chromatography, communoprecipitation, and newer approaches such as protein chip arrays, hold much promise in the detection of protein interactions, particularly in well-characterized organisms with sequenced genomes. However, each of these approaches attracts certain background proteins that can thwart detection and identification of true interactors. In addition, recombinant proteins expressed in Escherichia coli are also extensively used to assess protein-protein interactions, and background proteins in these isolates can thus contaminate interaction studies. Rigorous validation of a true interaction thus requires not only that an interaction be found by alternate techniques, but more importantly that researchers be aware of and control for matrix/support dependence. Here, we evaluate these methods for proteins interacting with DmsD (an E. coli redox enzyme maturation protein chaperone), in vitro, using E. coli subcellular fractions as prey sources. We compare and contrast the various in vitro interaction methods to identify some of the background proteins and protein profiles that are inherent to each of the methods in an E. coli system.