Activities

TMPC has declared its targets and target status to the PepcDB. Protocols for preparation of membrane proteins are being prepared, with specific efforts focusing on the newly proposed Target and Trial Status keyword “Membrane Protein Solubilized” and associated protocols.

We have already deposited the empty expression vectors used in this research into the PSI-Materials Respository. Additional empty expression vectors now in development in this project will be deposited when their utility has been established.

TMPC has begun planning with Membrane Protein Structural Biology Consortium, led by Michael Malkowski, to provide samples of membrane proteins prepared by cell-free translation for high-thoughput crystallization screening.

TMPC has partnered with the Chaperone-Enabled Biology and Structure project, led by Tony Kossiakoff, to design customized cell-free expression vectors that will be used in a novel “catch and release” method for rapid purification of membrane proteins stabilized by synthetic affinity binding proteins.

Current Research

Pilin Biosynthesis. PilD is a bifunctional protease/methyl transferase and enzyme. The Pseudomonas aeruginosa enzyme prepared by cell-free translation has both activities. This work is a collaboration of TMPC with Professor Katrina Forest (Department of Bacteriology, University of Wisconsin) and postdoctoral fellow Khaled Aly.

Sphingolipid Biosynthesis Pathway. This work is a collaboration of TMPC with Professor James Bangs (Department of Medical Microbiology and Immunology, University of Wisconsin). Cell-free translation was applied to elucidate the function of all isoforms of sphingolipid synthases from Trypanosoma brucei, T. cruzi, and Leishmania major. This work was undertaken according to our NEW COLLABORATOR PLAN, and was recently published. Since this founding work, we have continued to use cell-free translation and site-directed mutagenesis to establish residues essential for substrate selectivity, have identified detergents capable of solubilization, and piloted a scale-up purification protocol. These efforts set the stage for large-scale protein production to support crystallization screening. The sphingolipid biosynthesis pathway is found in most eukaryotes, and is structurally diverse. Several other integral membrane proteins from this pathway are of interest as targets for future studies.

Sigma-1 Receptor. This work is a collaboration of TMPC with Professor Arnie Ruoho (Department of Pharmacology, University of Wisconsin). Human sigma-1 receptor is an ~25 kDa protein found in the ER membrane of tissues of the endocrine, immune, and nervous systems. Sigma-1 receptor interacts with progesterone and testosterone, and other compounds including cocaine, amphetamines, hallucinogens, and others. Knockout mice exhibit changes in responses to stimuli, implicating sigma-1 in pain response, learning, and psychoses.

Publications

Beebe ET, Makino S-I, Nozawa A, Matsubara Y, Frederick RO, Primm JG, Goren MA, Fox BG (2010) Robotic large-scale application of wheat cell-free translation to structural studies including membrane proteins. N Biotechnol 28(3):239-49. PMID:20637905 / PMCID:PMC3035758

Blommel PG, Fox BG (2007) A combined approach to improving large-scale production of tobacco etch virus protease. Protein Expr Purif 55(1):53-68. PMID:17543538 / PMCID:PMC2047602

Blommel PG, Martin PA, Seder KD, Wrobel RL, Fox BG (2009) Flexivector cloning. Methods Mol Biol 498:55-73. PMID:18988018

Chae PS, Rasmussen SG, Rana RR, Gotfryd K, Chandra R, Goren MA, Kruse AC, Nurva S, Loland CJ, Pierre Y, Drew D, Popot JL, Picot D, Fox BG, Guan L, Gether U, Byrne B, Kobilka B, Gellman SH (2010) Maltose-neopentyl glycol (MNG) amphiphiles for solubilization, stabilization and crystallization of membrane proteins. Nat Methods 7(12):1003-8. PMID:21037590 / PMCID:PMC3063152

Chang Y, Mead D, Dhodda V, Brumm P, Fox BG (2009) One-plasmid tunable co-expression for mycobacterial protein-protein interaction studies. Protein Sci 18(11):2316-25. PMID:19760663 / PMCID:PMC2788286

Chang Y, Wesenberg G, Bingman CA, Fox BG (2008) In vivo inactivation of mycobacterial integral membrane stearoyl-CoA desaturase DesA3 by a C-terminal specific degradation process. J Bacteriol 190(20):6686-96. PMID:18723625 / PMCID:PMC2566213

Goren MA, Fox BG (2008) Wheat germ cell-free translation, purification, and assembly of a functional human stearoyl-CoA desaturase complex. Protein Expr Purif 62(2):171-8. PMID:18765284 / PMCID:PMC2586813

Goren MA, Fox BG, Bangs JD (2011) Amino acid determinants of substrate selectivity in the Trypanosoma brucei sphingolipid synthase family. Biochem 50(41):8853-61.PMID:21899277 / PMCID:PMC3212988

Goren MA, Nozawa A, Makino S, Wrobel RL, Fox BG (2009) Cell-free translation of integral membrane proteins into unilamelar liposomes. Methods Enzymol 463:647-73. PMID:19892197

Makino S-i, Goren MA, Fox BG, Markley JL (2010) Cell-free protein synthesis technology in NMR high-throughput structure determination. Methods Mol Biol 607:127-47. PMID:20204854

Sobrado P, Goren MA, James D, Amundson CK, Fox BG (2008) A Protein Structure Initiative approach to expression, purification, and in situ delivery of human cytochrome b5 to membrane vesicles. Protein Expr Purif 58(2):229-41. PMID:18226920 / PMCID:PMC2277500

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