Genetically encoded releasable photo-cross-linking strategies for studying protein-protein interactions in living cells
Genetically encoded releasable photo-cross-linking strategies for studying protein-protein interactions in living cells
复制标题
用于研究活细胞中蛋白质与蛋白质相互作用的基因编码可释放光交联策略
DOI：
10.1038/nprot.2017.090
复制
发表时间：
2017-10-01
期刊：
影响因子：
14.8
通讯作者：
Chen, Peng R.
中科院分区：
文献类型：
--
作者：
Yang, Yi;Song, Haiping;Chen, Peng R.
Although protein-protein interactions (PPIs) have crucial roles in virtually all cellular processes, the identification of more transient interactions in their biological context remains challenging. Conventional photo-cross-linking strategies can be used to identify transient interactions, but these approaches often suffer from high background due to the cross-linked bait proteins. To solve the problem, we have developed membrane-permeable releasable photo-cross-linkers that allow for prey-bait separation after protein complex isolation and can be installed in proteins of interest (POIs) as unnatural amino acids. Here we describe the procedures for using two releasable photo-cross-linkers, DiZSeK and DiZHSeC, in both living Escherichia coli and mammalian cells. A cleavage after protein photo-cross-linking (CAPP) strategy based on the photo-cross-linker DiZSeK is described, in which the prey protein pool is released from a POI after affinity purification. Prey proteins are analyzed using mass spectrometry or 2D gel electrophoresis for global comparison of interactomes from different experimental conditions. An in situ cleavage and mass spectrometry (MS)-label transfer after protein photo-cross-linking (IMAPP) strategy based on the photo-cross-linker DiZHSeC is also described. This strategy can be used for the identification of cross-linking sites to allow detailed characterization of PPI interfaces. The procedures for photo-cross-linker incorporation, photo-cross-linking of interaction partners and affinity purification of cross-linked complexes are similar for the two photo-cross-linkers. The final section of the protocol describes prey-bait separation (for CAPP) and MS-label transfer and identification (for IMAPP). After plasmid construction, the CAPP and IMAPP strategies can be completed within 6 and 7 d, respectively.
