Support is requested for Protein Production. While small-scale preparations of recombinant proteins using standard affinity purifications will be performed in the individual laboratories of the project, all projects require mg-amounts of highly purified recombinant and untagged proteins. The Core will provide technical and scientific support in two ways. First, it will optimize expression of proteins that either do not express at all or only at very low levels, including change of vectors, bacterial strains, co-expression of chaperones, or switching to an eukaryotic expression system. Second, the core will express and purify medium to large scale amounts of proteins using an array of conventional and affinity-based chromatographic methods. The core is equipped with a bacterial fermenter for large-scale expression, two state-of-the-art automated FPLC-systems, and a miniaturaized FPLC (SMART). An array of physico-chemical techniques will be used for initial characterization with respect to folding, oligomerization/aggregation and purity including CD-spectroscopy, multi-angle laser light scattering, and mass spectrometry. It is directd jointly by Drs. Fasshauer and Jahn, who will be assisted by M. Boeddener, an experienced Research Associate with several years of experience in all of the required techniques.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Program Projects (P01)
Project #
5P01GM072694-05
Application #
7797461
Study Section
Special Emphasis Panel (ZRG1)
Project Start
Project End
Budget Start
2009-04-01
Budget End
2010-03-31
Support Year
5
Fiscal Year
2009
Total Cost
$79,814
Indirect Cost
Name
University of Virginia
Department
Type
DUNS #
065391526
City
Charlottesville
State
VA
Country
United States
Zip Code
22904
Yavuz, Halenur; Kattan, Iman; Hernandez, Javier M et al. (2018) Arrest of trans-SNARE zippering uncovers loosely and tightly docked intermediates in membrane fusion. J Biol Chem 293:8645-8655
Liang, Binyong; Tamm, Lukas K (2018) Solution NMR of SNAREs, complexin and ?-synuclein in association with membrane-mimetics. Prog Nucl Magn Reson Spectrosc 105:41-53
Hussain, Syed Saad; Harris, Megan T; Kreutzberger, Alex J B et al. (2018) Control of insulin granule formation and function by the ABC transporters ABCG1 and ABCA1 and by oxysterol binding protein OSBP. Mol Biol Cell 29:1238-1257
Blackburn, Matthew R; Hubbard, Caitlin; Kiessling, Volker et al. (2018) Distinct reaction mechanisms for hyaluronan biosynthesis in different kingdoms of life. Glycobiology 28:108-121
Witkowska, Agata; Jablonski, Lukasz; Jahn, Reinhard (2018) A convenient protocol for generating giant unilamellar vesicles containing SNARE proteins using electroformation. Sci Rep 8:9422
Kiessling, Volker; Kreutzberger, Alex J B; Liang, Binyong et al. (2018) A molecular mechanism for calcium-mediated synaptotagmin-triggered exocytosis. Nat Struct Mol Biol 25:911-917
Nyenhuis, Sarah B; Cafiso, David S (2018) Choice of reconstitution protocol modulates the aggregation state of full-length membrane-reconstituted synaptotagmin-1. Protein Sci 27:1008-1012
Kreutzberger, Alex J B; Kiessling, Volker; Liang, Binyong et al. (2017) Asymmetric Phosphatidylethanolamine Distribution Controls Fusion Pore Lifetime and Probability. Biophys J 113:1912-1915
Tamm, Lukas K (2017) Special Issue on Liposomes, Exosomes, and Virosomes. Biophys J 113:E1
Jakhanwal, Shrutee; Lee, Chung-Tien; Urlaub, Henning et al. (2017) An activated Q-SNARE/SM protein complex as a possible intermediate in SNARE assembly. EMBO J 36:1788-1802

Showing the most recent 10 out of 76 publications