The Tandem Affinity Purification Technology To Create And Separation And Purification In The Tsc1 Protein Complex

With the complement of human genome project, a lot of scientist started to large scale study the functions of genes with high throughput technologies. For this purpose, proteomics is arisen. Herein, we describe the establishment of tandem affinity purification in mammalian cells and its application in purification of TSC1 protein complexes. In controst to overexpression of bait proteins, the weak expression strategy is more suitable for proteome scale research. It involves in intergrating the tagged bait gene into the genome of mammalian cells with retroviruses and expressing the protein under physiological conditions by modifying the multiplicity of infection (MOI).With lower expression levels of the tagged proteins, we need to culture more cells to get sufficient proterins for purification, visualization and identification. We solved this problem by using Cell Factories which can be used to large-scalely culture adherent cells. We finally used the following process of purification : tandemly purification of interesting protein complex (include negative control)â†'separated by SDS-PAGEâ†'stained with Commasie Brilliant Blueâ†'cut protein bands specific for sampleâ†'LTQ-MS.Tuberous sclerosis complex (TSC) is a benign tumor identified in numerous tissues such as brain, kidney and lung. Mutations in either TSC1 or TSC2 gene lead to TSC. These two genes are reported to form a complex in vivo. With the TAP-MS strategy described above, we started with 1×10~9 cells and completed the purification of proteins associated with TSC1. Under stringent purification and identification conditions, 150 proteins were found potentially interacting with TSC1. Two proteins among the list, TSC2 and DOCK7, have been reported to associate with TSC1 directly , which indicating that we can efficiently purify TSC1 protein complexes by TAP-MS strategy.Bioinformatics and biochemical methods were used to evaluate and validate the reliability of the results. Picasso (which is developed by bioinformatics group of our lab) analysis showed that the positive rate is around 37%. Six randomly chosen proteins can associate wich TSC1 in co-immunoprecipitation assay. Moreover, both comparing with microarray data and cellular localization analysis implied that the dataset is reliable.Then, we found that proteins with coiled-coil domain in the dataset are selectively enriched for 2.7 times, which might interact directly with TSC1. We also found a group of proteins (nearly 10%) are involved in neuronal diseases. Relationships of several selected proteins with TSC1 are further discussed.To sum, we examined the human TSC1 complex with tandem affinity purification under physiological status. Our study reveals complexity of protein components in the TSC1 complex(es) uncovers a number of previously unknown binding partners of TSC1 and provides a platform for further investigating biological function and molecular mechanisms of TSC complex (es).