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Tissue- and isoform-specific protein complex analysis with natively processed bait proteins.
Journal of Proteomics ( IF 3.3 ) Pub Date : 2020-08-24 , DOI: 10.1016/j.jprot.2020.103947
Tina Beyer 1 , Franziska Klose 1 , Anna Kuret 2 , Felix Hoffmann 1 , Robert Lukowski 2 , Marius Ueffing 1 , Karsten Boldt 1
Affiliation  

Protein-protein interaction analysis is an important tool to elucidate the function of proteins and protein complexes as well as their dynamic behavior. To date, the analysis of tissue- or even cell- or compartment-specific protein interactions is still relying on the availability of specific antibodies suited for immunoprecipitation. Here, we aimed at establishing a method that allows identification of protein interactions and complexes from intact tissues independent of specific, high affinity antibodies used for protein pull-down and isolation. Tagged bait proteins were expressed in human HEK293T cells and residual interactors removed by SDS. The resulting tag-fusion protein was then used as bait to pull proteins from tissue samples. Tissue-specific interactions were reproducibly identified from porcine retina as well as from retinal pigment epithelium using the ciliary protein lebercilin as bait. Further, murine heart-specific interactors of two gene products of the 3′,5′-cyclic guanosine monophosphate (cGMP)-dependent protein kinase type 1 (cGK1) were investigated. Here, specific interactions were associated with the cGK1α and β gene products, that differ only in their unique amino-terminal region comprising about 100 aa. As such, the new protocol provides a fast and reliable method for tissue-specific protein complex analysis which is independent of the availability or suitability of antibodies for immunoprecipitation.

Significance

Protein-protein interaction in the functional relevant tissue is still difficult due to the dependence on specific antibodies or bait production in bacteria or insect cells. Here, the tagged protein of interest is produced in a human cell line and bound proteins are gently removed using SDS. Because applying the suitable SDS concentration is a critical step, different SDS solutions were tested to demonstrate their influence on interactions and the clean-up process. The established protocol enabled a tissue-specific analysis of the ciliary proteins lebercilin and TMEM107 using pig eyes. In addition, two gene products of the 3′,5′-cyclic guanosine monophosphate (cGMP)-dependent protein kinase type 1 showed distinct protein interactions in mouse heart tissue. With the easy, fast and cheap protocol presented here, deep insights in tissue-specific and functional relevant protein complex formation is possible.



中文翻译:

用天然加工的诱饵蛋白进行组织和同工型特异性蛋白复合物分析。

蛋白质-蛋白质相互作用分析是阐明蛋白质和蛋白质复合物的功能及其动态行为的重要工具。迄今为止,组织或什至细胞或区室特异性蛋白质相互作用的分析仍依赖于适用于免疫沉淀的特异性抗体的可用性。在这里,我们旨在建立一种方法,该方法可以从完整的组织中识别蛋白质相互作用和复合物,而与用于蛋白质下拉和分离的特异性高亲和力抗体无关。标记的诱饵蛋白在人HEK293T细胞中表达,残留的相互作用因子通过SDS去除。然后将所得的标签融合蛋白用作诱饵,以从组织样品中提取蛋白。使用睫毛蛋白lebercilin作为诱饵,可从猪视网膜以及视网膜色素上皮中重现组织特异性相互作用。另外,研究了3',5'-环鸟苷单磷酸(cGMP)依赖性蛋白激酶1型(cGK1)的两个基因产物的小鼠心脏特异性相互作用子。在这里,特定的相互作用与cGK1α和β基因产物有关,它们的区别仅在于其独特的氨基末端区域(约100个氨基酸)。这样,新协议为组织特异性蛋白质复合物分析提供了一种快速可靠的方法,而该方法与免疫沉淀抗体的可用性或适用性无关。研究了5'-环鸟苷一磷酸(cGMP)依赖性蛋白激酶1型(cGK1)。在这里,特定的相互作用与cGK1α和β基因产物有关,它们的区别仅在于其独特的氨基末端区域(约100个氨基酸)。这样,新协议为组织特异性蛋白质复合物分析提供了一种快速可靠的方法,而该方法与免疫沉淀抗体的可用性或适用性无关。研究了5'-环鸟苷一磷酸(cGMP)依赖性蛋白激酶1型(cGK1)。在这里,特定的相互作用与cGK1α和β基因产物有关,它们的区别仅在于其独特的氨基末端区域(约100个氨基酸)。这样,新协议为组织特异性蛋白质复合物分析提供了一种快速可靠的方法,而该方法与免疫沉淀抗体的可用性或适用性无关。

意义

由于对特定抗体的依赖性或细菌或昆虫细胞中诱饵的产生,功能相关组织中的蛋白质-蛋白质相互作用仍然很困难。在这里,标记的目的蛋白在人细胞系中产生,结合的蛋白使用SDS轻轻去除。由于应用合适的SDS浓度是关键步骤,因此对不同的SDS溶液进行了测试,以证明它们对相互作用和净化过程的影响。建立的协议可以使用猪眼对睫状蛋白lebercilin和TMEM107进行组织特异性分析。另外,3',5'-环鸟苷单磷酸(cGMP)依赖性蛋白激酶1型的两个基因产物在小鼠心脏组织中表现出不同的蛋白相互作用。通过此处介绍的简单,快速和廉价的协议,

更新日期:2020-10-13
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