stract (Official) 1) Bacterial Protein Expression Core (Hill. Director: Schubert. Manager) Overview The Bacterial Protein Expression Core is located on the third floor of the EEJMRB at the University of Utah In close proximity to the labs of Hill, Sundquist, and Kay. It is managed by Heidi Schubert and will be staffed by two Center-funded technicians. The facility provides efficient construction of expression vectors, testing of protein expression and solubility, protein purification, and characterization. We have established a synergistic relationship with the Utah Molecular Hematology Protein Expression Core for the cost effective production of recombinant TEV protease and Pfu polymerase, and employ services of the University of Utah core facilities in mass spectrometry, oligonucleotide synthesis, N-terminal protein sequencing, and DNA sequencing. The Bacterial Protein Expression Core facilities are integrated with operations of the Eukaryotic Protein Expression Core for bioinformatics, cloning, and protein purification, and there is also a close association with protein structure determination efforts in X-ray and Protein NMR cores. This core will therefore facilitate structural and biochemical studies on targets identified by the Center members pursuing questions of HIV/Host biology. In general, the methods used in this Core are standard, but have been adapted to maximize efficiency on a scale appropriate for the targeted, but ambitious, scope of this application. The basic instrumentation and protocols are already in place and will be expanded to match increased demand if the Center is funded. The increased throughput will be achieved by efficient database management, consolidated bioinformatics resources, use of technical staff, and standardization and refinement of protocols. The approaches are an extension of the methodology that has supported structural and biochemical studies by the Hill and Sundquist labs (see biosketches for publications). Our initial target capacity is an average of 24 new expression vectors constructed per week, although the actual rate will fluctuate in response to demand. This level of throughput will allow aggressive approaches to problems, such as optimizing N- and C-termini or making surface mutations to produce crystallizable constructs. If demand exceeds capacity, we can add personnel/instrumentation/supplies on a charge-back basis.
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