TMPC is organized into research teams which address each of the constituent tasks of structural genomics. We also work closely with collaborators whose expertise in the function of membrane protein targets provides unique biological insight needed to advance our structure determination efforts and to guide interpretation and dissemination of results. Complementary cell-free and cell-based translation methods are currently being used to prepare proteins in the target classes of sphingolipid synthases, sigma-1 receptor, fatty acid desaturases, and pilin assembly complexes for functional studies and structure determination.
We are developing cell-free translation, which utilizes ENDEXT® technology, as a versatile method for studying membrane protein structure and function. TMPC’s experimental platform is based on establishing predictive behaviors for membrane proteins using free translation in small-scale (50 µL to 1.2 mL). Robotic translation has an advantage of simplicity and reproducibility in the provision of material used for further research and protocol development. When appropriate expression and solubilization conditions are identified, purification protocols are developed in small-scale, and then the work is transferred to different robots whose operational volume (1.2 mL to 20 mL) better supports structure determination or expanded biological studies.
The suite of cell-free translation robots in use at TMPC were developed by CellFree Sciences Co., Ltd. (Yokohama, Japan). These instruments, and the personnel and expertise to operate them, were assembled during PSI-1 and PSI-2. No other freely accessible site exists worldwide with this capability. We believe these robots will be a valuable resource for the PSI:Biology Network.
Other research infrastructure available at the TMPC and associated laboratories includes Sesame LIMS, Maxwell-16 Automated Purification System, AKTA Purification Systems, Shimadzu HPLC System with UV-Vis and fluorometric detection customized for use with detergents, TTP Lab Tech's Mosquito nanoliter crystallization robot, CrystalFarm Imaging System (Bruker AXS) with CrystalFarm Pro Data Management System, and routine access to GM/CA-CAT, SER-CAT, and LS-CAT at Advanced Photon Source.
Crystallization screening is being carried out in the TMPC facilities and at PSI:Biology Membrane Protein Structural Biology Consortium, led by Dr. Michael Malkowski.
Technologies at PSI SBKB Technology Portal | See All at Tech Portal
Annotation and Function
- Amino acid determinants of substrate selectivity in the Trypanasoma brucei sphingolipid synthase family. (Read at Tech Portal)
- Cell-free synthesis and functional characterization of sphingolipid synthases from parasitic trypanosomatid protozoa. (Read at Tech Portal)
- Differential scanning flourimetry for identification of protein ligands and stabilizing conditions at TMPC. (Read at Tech Portal)
- A combined approach to improving large-scale production of tobacco etch virus protease. (Read at Tech Portal)
- Cell-free translation of integral membrane proteins into unilamelar liposomes. (Read at Tech Portal)
- Customized expression vector platform. (Read at Tech Portal)
- In vivo inactivation of mycobacterial integral membrane stearoyl-CoA desaturase DesA3 by a C-terminal specific degradation process. (Read at Tech Portal)
- One-plasmid tunable co-expression for mycobacterial protein-protein interaction studies. (Read at Tech Portal)
- The use of PIPE cloning at TMPC. (Read at Tech Portal)
Protein Expression and Purification
- A Protein Structure Initiative approach to expression, purification, and in situ delivery of human cytochrome b5 to membrane vesicles. (Read at Tech Portal)
- Activity and topology determination of in vitro expressed membrane proteins. (Read at Tech Portal)
- Cell-free production of integral membrane aspartic acid proteases reveals zinc-dependent methyltransferase activity of the Pseudomonas aeruginosa prepilin peptidase PilD. (Read at Tech Portal)
- Co-translation of integral membrane proteins (IMP) with membrane scaffold proteins (MSP), also known as nanodiscs. (Read at Tech Portal)
- Detergent solubilization screening for membrane proteins expressed in E. coli. (Read at Tech Portal)
- Development of detergent arrays for membrane protein compatibility screening. (Read at Tech Portal)
- Expression and purification of bacteriorhodopsin using Protemist XE with detergents. (Read at Tech Portal)
- Function of Shaker potassium channels produced by cell-free translation upon injection into Xenopus oocytes. (Read at Tech Portal)
- Optimization of membrane protein production in E. coli and robotic purification. (Read at Tech Portal)
- Robotic large-scale application of wheat cell-free translation to structural studies including membrane proteins. (Read at Tech Portal)
- Structural genomics methods applied to production of the monotopic membrane protein human cytochrome b5 and in situ delivery to liposomes. (Read at Tech Portal)
- Variations in linker region of MBP-S1R fusion protein dramatically influences the yield of highly purified, active membrane receptor. (Read at Tech Portal)
- Wheat germ cell-free translation, purification, and assembly of a functional human stearoyl-CoA desaturase complex. (Read at Tech Portal)
- Maltose–neopentyl glycol (MNG) amphiphiles for solubilization, stabilization and crystallization of membrane proteins. (Read at Tech Portal)