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Missions Methodologies

Specific Aims for Research Cores

Protein Production Core:
  Pyrococcus furiosus ( more details and people)
  • Construct a Protein Production Facility containing high through (HTP)-Cloning, HTP-Expression and HTP-Protein Production Modules.
  • Clone the approximately 2,800 single (s) and multiple (m) ORFs derived from the P. furiosus genome in the HTP-Cloning Module
  • Develop five different sets of Expression Protocols (I-V) to produce all classes of proteins in the HTP-Expression Module in a FUNCTIONAL form
  • Compare the success in expressing multi-subunit proteins (from mORFs) with the expression of their individual subunits (from sORFs)
  • Determine the actual success in expressing sORFs and mORFs using the Expression Protocols with that predicted from ORF analyses
  • Ultimately develop a model system based on ORF analysis for the successful expression of any gene
  • Produce a minimum of 30 proteins per week (~ 5mg each) at the analytical level and at least 5 proteins per week (~ 50mg each) on the preparative scale in the HTP-Protein Production Module
  Caenorabditis elegans (more details and people)
  • Develop high throughput robotic cloning and expression of C. elegans proteins using novel Gateway vectors.
  • Apply a high throughput protocol for the purification of C. elegans protein with the rate of over 40 proteins per week (~1 mg each)
  • Expand protein refolding methodologies from inclusion bodies expressed in E. Coli as a cost-effective way of protein production
  Human (more details and people)
  • Prescreen available Mammalian Gene Collection cDNAs for suitable targets.
  • Clone target cDNAs into custom pTrcHis-TOPO vector with the goal of producing ~30 proteins per week.
  • Nonexpressing/nonpurifying proteins will be subjected to secondary protocols as time allows.
NMR Core: (more details and people)
  • Establish automated classification protocol identifying proteins most suited for structure determination by NMR and by X-ray crystallography
  • Develop automated condition optimization protocol for structure determination by NMR methods.
  • Develop high throughput protocol for back-bone structure determination by NMR
  • Determine protein structures with possible new fold, but which could not be crystallized
  • Aim to produce 1-3 back-bone NMR structures per week
X-ray Crystallography Core: (more details and people)
  • Develop a robotic high throughput crystal screening (HTP-CS) system with a goal of producing 1,000 trials per hour using 300 micrograms of protein sample
  • Develop automated synchrotron data collection using sample-placement robot to increase data throughput by a factor of three to five
  • Develop on-the-fly data evaluation and map calculation capabilities at synchrotron sites to improve data collection efficiency
  • Avoid the cost associated with the preparation of Se-Methionine labeled proteins in Se-MAD method by exploiting recent developments of de novo phase estimation using the anomalous scattering signal of sulfur and metal atoms inherently present in the protein by single-wavelength anomalous scattering methods
  • Aim to produce 3-6 refined high resolution protein structures per week
Bioinformatics Core: (more details and people)
  • Develop software and database tools to track experimental results
  • Streamline integration and correlation of information from disparate sources
  • Regularly update important databases related to our selected target proteins
  • Automatically disseminate information to research community
  • Carry out high-throughput computation by use of a multi-processor computer cluster to analyze biological information, predict and model protein structures, find crystal and NMR structure solutions, and validate structures on a large scale
Crystallomics Core: (more details and people)  
  • Prototype PSI-2 Large-Scale Production Center formed in September 2003
  • Use crystal-directed protein production
  • Use carefully selected technologies with an industrial production-oriented philosophy to build high-throughput capabilities in cloning, expression, protein purification and crystallization
  • Combine techniques with advanced high-throughput structure determination pipelines developed by the Crystallography Core and bioinformatics tools to form a prototype Production Center