Da Yu Protein Sciences – Protein Folding
Many recombinant proteins overexpressed in E. coli are produced in an inactive form as insoluble inclusion bodies. Sometimes inclusion body formation can be avoided by altering the expression plasmid, the E. coli strain, growth conditions or the use of novel approaches such as our Perseus™ Expression Technology. When these measures fail, or they are not in alignment with a CMC development strategy, the only alternative is to refold the protein from solubilized inclusion bodies.
Protein refolding from inclusion bodies is dependent on many factors including, but not limited to, the following:
- Protein concentration
- Refolding pH
- Redox environment
- Solution polarity
- Buffers and excipients
Da Yu Protein Sciences utilizes a fractional factorial design of experiments (DOE) approach to protein folding based on the protein’s theoretical profile. This strategy is intended to yield at least some protein folding from which further experiments can be designed to optimize the refolding conditions for an individual protein. We can monitor proper protein folding by a variety of procedures. A protein’s function is related to its structure. Thus, detecting a protein’s function is indicative of proper structure. At Da Yu Protein Sciences we have the capability to monitor a protein’s functional properties by a variety of means. If a functional assay is not available, Da Yu Protein Sciences also has the cababilities to monitor properly-folded proteins by a variety of biophysical techniques including Circular Dichroism Spectroscopy and UV Derivative Spectroscopy. For proteins at low concentrations that are not amenable to these techniques we also have the capability of detecting a protein’s properly folded structure by melting temperature (Tm) using Differential Scanning Fluorimetry or monitoring changes in the binding of extrinsic fluorescent probes.
Da Yu Protein Sciences has experience in refolding many recombinant proteins derived from E. coli. We have a library of more than 100 buffers and excipients available for client protein refolding experiments. We also have the means of producing sufficient amounts of washed inclusion bodies for these experiments utilizing our capabilities in E. coli Fermentations
Clients interested in discussing our protein folding development services are encouraged to Contact Us.