Posted: November 26, 2015
Affinity tags are highly efficient tools for purifying proteins from crude extracts. However, most affinity purification elusion schemes require a change in buffer condition, such as addition of imidazole or a decrease in pH. This change in buffer conditions may cause some proteins that are non-specifically bound to the resin to elute as contaminants and damage or denature the protein of interest. In most cases, subsequent dialysis or desalting is required to exchange the purified protein from elution buffer into a more suitable buffer for storage or downstream analysis resulting in a tradeoff between high purity or high yield. In addition, further downstream processing is required to determine protein yield which wastes precious protein and creates additional steps in the automation process.
Researchers at the University of Alberta have developed a photocleavable recombinant fusion tag for light-induced elution of proteins from a purification resin during affinity purification. This new elution scheme ensures that only the protein of interest is eluted and avoids the compromise between high purity and high yield as well as the potential damage caused by some elution buffers. The tag undergoes photocleavage and spontaneous dissociation within a few minutes leaving a small peptide associated with the protein of interest. This peptide provides a unique visible wavelength spectral handle that can be used to determine the concentration of the protein of interest facilitating downstream applications by making the protein concentration determination a trivial and consistent procedure that works identically for any protein of interest. Furthermore, the unique chemical reactivity of the chromophore may also be useful for downstream detection and assay methods such as chemical modifications and labeling.
- Amendable to high-throughput automation.
- High protein purity, high yield of intact protein of interest.
- Unique spectral handle for determining protein concentration.
- Adaptable to downstream modifications and/or labeling.
The Invention represents a valuable opportunity for uses ranging from laboratory-scale production (micrograms) to process scale (gram-kilogram) production.
Technology Management Group
TEC Edmonton – University of Alberta