Screening For The Proteins Interacting With Stratifin In Human Colon Cancer Stem Cells By Yeast Two-hybrid Assays

BackgroundIn the previous study, we isolated SW1116csc from SW1116cells, and identified differentially expressed proteins by tandem mass spectrometry (MALDI-TOF/TOF) between them. The expression of Stratifin was almost30-fold increased in SW1116csc compared with that in SW1116cells. In the current research, we looked for proteins interacting with Stratifin in human colon cancer stem cells. Materials and methodsHuman colon cancer stem cells (SW1116csc cells) were isolated from the human colon cancer cell line (SW1116) and cultured in serum-free medium. Based on the MATCHMAKER GAL4yeast two-hybrid system3, total RNA was extracted for reverse transcription and synthesis of cDNA. cDNA was transformed into E. coli DH10B and constructed cDNA primary library, and then recombined with pGADT7to form the library plasmid. Recombined Stratifin gene with pGBKT7by restriction enzyme cutting site of Sfil, and transformed into E. coli Top10to form the BD plasmid of pGBKT7-SFN. Kanamycin selected positive clones, and the enzyme cutting extracted the plasmids that had been identified the size of inserted-pieces. The pGADT7-SW1116csc and the pGBKT7-SFN were transformed into yeast strain AH109, and the bait plasmid was tested for toxicity and self-activating effect. Interacting proteins of Stratifin and cDNA library of SW1116csc were screened by Yeast two-hybrid system. Screened for His3+/LacZ+/Ade2+positive colonies and analyzed the sequences.ResultsThe size of inserted-pieces of constructed-pGADT7-SW1116csc was about1.5Kbp after PCR, and pGBKT7-SFN resulted in pieces which were larger than1.0Kbp by restriction enzyme digestion. Both of them were qualified. Both pGBKT7-SFN and pGADT7-SW1116csc were transformed into yeast strain AH109without toxicity and self-activating effect. We screened7His3+/LacZ+/Ade2+positive colonies which could interact with Stratifin using yeast two-hybrid mating. Compared the sequences with that of other proteins in NCBI, and we got4related proteins, which were BAD, MPRIP, CHRM17and TMEM45B.ConclusionWe constructed successfully the human colon cancer stem cells cDNA plasmid of pGADT7-SW1116csc which was correctly expressed in yeast. And we screened4proteins which could interact with Stratifin in human colon cancer stem cells, including BAD、 MPRIP、CHRM17and TMEM45B. Our study sheds light on the regulatory mechanism of Stratifin to proliferation and differentiation of colon cancer stem cells. This study will lay foundation for the further research.