Core Capabilities &Approach: The Protein Core has accumulated and demonstrated the required expertise in recombinant protein expression and purification utilizing bacterial and insect cell systems. The protein expression platform will be centered on 6X-His and/or Strep tagged proteins, with or without other fusion partners, such as GB1, thioredoxin, NusA, MBP, and GST. Fusion constructs will contain TEV or thrombin cleavage sites for down-stream removal of the fusion protein. For each expression vector variant, multiple conditions will be tested to achieve optimal expression and yield: host, temperature, induction time, etc. For production in E. coli, the optimization approaches will include: 1) incremental truncation/extension of the coding sequence to define minimal folding domains, 2) mutagenesis or in vitro evolution of the coding sequence in the context of preserved biological function, and 3) a combination of) and 2). This approach will result in large numbers of constructs, requiring high-throughput cloning, expression, and solubility screening. These tasks have been successfully handled by the Core and yielded difficult proteins such as Vpr and Vpx in forms amenable to structure determination. Despite these efforts, production of soluble virus-derived or cellular proteins in E. coli may fail for some targets. In these cases, targets will be expressed in insect cells. The high-throughput methodologies for cloning, expression screening, and protein production in eukaryotic systems have been established and utilized for expression and purification of Trimsa, DCAFi, and DDBi. Purified proteins will be characterized by static multi-angle light scattering to assess their quaternary state, LC-ESI-TOF mass spectrometry to confirm purity, NMR isotope labeling efficiency and selenomethionine (Se-Met) labeling, before submitting samples for NMR and X-ray crystallography. Labeling protocolsf^39-242) are well-established and routinely used. For proteins purified from eukaryotic expression systems, post-translational modification will be analyzed using ESI-TOF and other mass spectrometry methods. To determine protein stability, circular dichroism, fluorescence spectroscopy, and differential scanning calorimetry will be performed as a function of temperature and chaotropic agents. iii. Project Component: The Protein Core has been the main driving force in the PCHPI's studies of Vpr and DCAFif48,86) and will continue to play a primary role in these studies (Ps).

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Specialized Center (P50)
Project #
2P50GM082251-06
Application #
8528175
Study Section
Special Emphasis Panel (ZRG1-AARR-K (50))
Project Start
Project End
Budget Start
2012-09-30
Budget End
2013-07-31
Support Year
6
Fiscal Year
2012
Total Cost
$456,958
Indirect Cost
$110,718
Name
University of Pittsburgh
Department
Type
DUNS #
004514360
City
Pittsburgh
State
PA
Country
United States
Zip Code
15213
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