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Laboratory of Membrane Transport and Cell Metabolism

Published :2017-06-23 11:06:21

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 metabolism and structural andbiological research of potential drug target. The methods and Technology we useare down below:

1. Protein expression and purification

The crucial parts include heterogeneousexpression of recombinant protein via different systems and getting stablehomogeneous protein. We have set up E.coli,yeast, insect and mammalian cell expression systems. Through overexpression ofmembrane protein and soluble protein with several expression 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- expression,  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.


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