Laboratory of Membrane Transport and Cell me-x-tabolism
Principal Investigator: DongDeng
Method and Technology
The lab focuses on molecularmachineries of protein transporter, assembly and dynamic machineries of keyprotein complex in signaling pathway related to me-x-tabolism and structural andbiological research of potential drug target. The methods and Technology we useare down below:
1. Protein ex-x-pression and purification
The crucial parts include heterogeneousex-x-pression of recombinant protein via different systems and getting stablehomogeneous protein. We have set up E.coli,yeast, insect and mammalian cell ex-x-pression systems. Through overex-x-pression ofmembrane protein and soluble protein with several ex-x-pression vectors, such aspET, pPICZ, pFastbac, pCAG, and by using affinity chromatography (His tag, GSTtag, strep tag...), ion exchange chromatography and size exclusion chromatography,we could get high purity protein. Till now, we have already prepared someprotokaryotic/eukaryotic protein samples for crystallization and biochemicalexperiments. The lab has two high performance liquid chromatography systems(AKTA Pure, GE Healthcare) and several prepacked columns for proteinpurification and identification.
2. Protein complexreconstruction in vitro and biochemical research
Reconstructing protein complexin vitro has huge significances for studying the protein interactions. The lab applies co- ex-x-pression, pull down in vitro/vivo,extracting from natural organization and other methods to protein preparationfor the following research. We also use ITC, SPR and the other biophysicsmethods to measure the interaction between protein and micro molecular,polypeptide or other protein. In addition, we analyze protein biochemicalproperties by combining protein engineering and other methods together.
3. Crystallization of protein
There are several primarytechnologies which can be used in protein structural analysis, such as cryo-EM,X-ray and MRI. We apply vapor diffusion , including hanging drop and sittingdrop, and lipid cubic phase (LPC) for crystal screening and optimization withthe support of Mosquito and Gryphon from public laboratory platform.