The Expression And Crystallization Screening Of TBC1D15from Shark Liver

APSL is a hepatocyte stimulating cytokine which is isolated and purified from the liver ofchiloscyllium plagiosum，and has a potential therapeutic effect on the treatment of diabetes.Recently researches have showed that APSL has a good effect on anti-type2diabetes，and caneffectively improve the complications of diabetes. So it provided a theoretical basis for thedevelopment of a new anti-type2diabetic oral drug. However，it is difficult to develop this newdrug because the action mechanism and the drug target of APSL is still unknown. So the crystalstructure analysis of APSL can help us to understand the action mechanism and find the drugtarget ofAPSL.We successfully expressed and purified APSL and the fusion protein MBP-APSL. Theprotein crystallization experiments showed that APSE protein and MBP-APSL fusion proteincould not be crystallized because they all formed precipitation in all kinds of conditions. Atlast，we found that theAPSL was not a complete protein by bioinformatics analysis.We screened an ORF which contained2133bp from the chiloscyllium plagiosumregenerated liver cDNA library by comparing with APSL gene sequence. Bioinformatics analysisshowed that it had high homology with the TBC1D15protein，and the APSL gene was locatedin the unknown function domain DUF3548of N-terminal. So we speculated that the protein wegot from shark regenerative liver was a new TBC1D15protein. Then we expressed and purifiedTBC1D15protein through prokaryotic and eukaryotic expression system. The crystallizationconditions screening is still in progress.In order to further ensure the protein we purified was TBC1D15protein，we conductedRab-GAP activity experiment. As the Rab7a protein can be specifically identified by TBC1D15protein，we got the Rab7a-Q67L and Rab7a-T22N gene from the Rab7a-wt gene by overlapPCR. And then expressed and purified the fusion protein Biotin-MBP-Rab7a-wt/Q67L/T22N todetect whether TBC1D15had Rab-GAP activity. The alphascreen experiment showed that thepurified TBC1D15had Rab-GAP activity. This showed that the protein we purified from sharkregenerative liver was TBC1D15protein.Nowadays, there is still no report about the crystal structure of TBC1D15protein. In thisstudy, we firstly found TBC1D15from shark regenerative liver and expressed and purified itsuccessfully, which laid a foundation for further analysis of the crystal structure of TBC1D15.