User:Timothy L. Foley/Notebook/refolding matrix
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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.
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.