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Protein Production Core:
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Pyrococcus furiosus (
more details and people) |
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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
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Caenorabditis elegans
(more details and people) |
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- 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
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Human
(more details and people) |
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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.
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NMR Core:
(more details and people) |
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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
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X-ray Crystallography Core:
(more details and people) |
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- 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
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Bioinformatics Core:
(more details and people) |
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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
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Crystallomics Core:
(more details and people) |
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- 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
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