User:Timothy L. Foley/Notebook/refolding matrix

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==Project Description==
==Project Description==
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The heterologous production of soluble, active enzymes/proteins in E. coli is a significant hurdle to functional gene analysis in the postgenomic era. A number of reasons can account for the inability to convince E. coli to accumulate foreign polypeptides, but most of these (in my experience) can be resolved by whole gene synthesis in codon usage adjusted for bias of the host.  
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The heterologous production of soluble, active enzymes/proteins in ''Escherichia coli'' is a significant hurdle to functional gene analysis in the postgenomic era. A number of reasons can account for the inability to convince ''E. coli'' to accumulate foreign polypeptides, but most of these (in my experience) can be resolved by whole gene synthesis in codon usage adjusted for bias of the host.  
The new bottleneck imposed is the ability (or lack there of) to identify culture conditions that encourage the accumulation of soluble protein at the time of expression. As such, protein refolding subsequent to purification from inclusion bodies may provide a viable avenue to access appreciable quantities of polypeptides in their properly folded state.  
The new bottleneck imposed is the ability (or lack there of) to identify culture conditions that encourage the accumulation of soluble protein at the time of expression. As such, protein refolding subsequent to purification from inclusion bodies may provide a viable avenue to access appreciable quantities of polypeptides in their properly folded state.  
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There are no methodological approaches to resolve conditions that refold a protein based on primary sequence, but the [http://refold.med.monash.edu.au/| REFOLD] database has recently been compiled to catalog refolding data. Recently some commericially available matricies of buffer conditions have become available, including the [http://www.merck-chemicals.com/life-science-research/protein-refolding-kits/c_L9Cb.s1ORVUAAAEjYBp9.zLX| iFold] kits from Novagen,[http://www.hamptonresearch.com| FoldIt] kit from Hampton Research (crystal people, must be good), and [http://www.piercenet.net| Pro-Matrix]from Pierce/Thermofisher. However, their relatively high cost (e.g. iFOLD kits are $445, and provide buffers for a single 96 well plate experiment with a single protein at a single concentration, and are available in series 1-3) prohibit their use in the current funding climate.
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There are no methodological approaches to resolve conditions that refold a protein based on primary sequence, but the [http://refold.med.monash.edu.au/ REFOLD] database has recently been compiled to catalog refolding data. Recently some commericially available matricies of buffer conditions have become available, including the [http://www.merck-chemicals.com/life-science-research/protein-refolding-kits/c_L9Cb.s1ORVUAAAEjYBp9.zLX iFold] kits from Novagen,[http://www.hamptonresearch.com FoldIt] kit from Hampton Research (crystal people, must be good), and [http://www.piercenet.net Pro-Matrix] from Pierce/Thermofisher. However, their relatively high cost (e.g. iFOLD kits are $445, and provide buffers for a single 96 well plate experiment with a single protein at a single concentration, and are available in series 1-3) prohibit their use in the current funding climate.
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I will detail here my experiments to develop some homecooked matricies to support some inhouse structural genomics endeavors during my last months in graduate school. These protocols will include
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I will detail here my experiments to develop some homecooked buffer matricies to support an structural genomics endeavors during my last months in graduate school.
==Notes==
==Notes==

Revision as of 22:42, 13 October 2009

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Project Description

The heterologous production of soluble, active enzymes/proteins in Escherichia coli is a significant hurdle to functional gene analysis in the postgenomic era. A number of reasons can account for the inability to convince E. coli to accumulate foreign polypeptides, but most of these (in my experience) can be resolved by whole gene synthesis in codon usage adjusted for bias of the host.

The new bottleneck imposed is the ability (or lack there of) to identify culture conditions that encourage the accumulation of soluble protein at the time of expression. As such, protein refolding subsequent to purification from inclusion bodies may provide a viable avenue to access appreciable quantities of polypeptides in their properly folded state.

There are no methodological approaches to resolve conditions that refold a protein based on primary sequence, but the REFOLD database has recently been compiled to catalog refolding data. Recently some commericially available matricies of buffer conditions have become available, including the iFold kits from Novagen,FoldIt kit from Hampton Research (crystal people, must be good), and Pro-Matrix from Pierce/Thermofisher. However, their relatively high cost (e.g. iFOLD kits are $445, and provide buffers for a single 96 well plate experiment with a single protein at a single concentration, and are available in series 1-3) prohibit their use in the current funding climate.

I will detail here my experiments to develop some homecooked buffer matricies to support an structural genomics endeavors during my last months in graduate school.

Notes

  • A short notebook including my homemade matricies. Some of the discussion will require background education in statistical analysis and experimental design, which is taught by the psychology department here at UCSD. I took Biological Statistics (BIO 215) with Jim Sumich at Grossmont College (El Cajon, CA) as an undergraduate pretransfer student, and this provided a knowledgebase and software set sufficient to try these experiments.
  • THAT BEING SAID: If you have an exquisite background in experimental design and statistical analysis, please feel free to comment on my approaches to aid future scientists in applying the findings to their own research.
  • You will also need the JMP In software set to complete this work. I will use JMP IN v. 4.0.4 to set up my experiments and analyze the results. This is available (internally @ UCSD) for $40/year annual license, but I will take advantage of this because I am too close to graduation to warrant purchasing the software with personal funds.

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