A method to purify proteins with light
Proteins play a key role in the life sciences — from basic research and biotechnological applications to the development and manufacturing of pharmaceuticals. Scientists at the Technical University of Munich (TUM) have developed a method that relies on physics rather than conventional chemistry to obtain the proteins that are needed for this purpose. Using short-wave, UV light invisible to humans, they have succeeded in purifying proteins from cell extracts or cultures. This technique is more efficient and gentler than previous methods.
Scientists engaged in molecular biology or molecular medicine require proteins in their pure form for various purposes, to serve for investigation or as active substances. Such proteins are isolated from natural sources or produced with the help of genetically modified cells.
To this end, affinity chromatography has been the method of choice since 50 years. In this method, the cell extract or culture medium is passed through a chromatography column filled with a porous carrier material. The target protein is bound to this carrier material and separated from other proteins and impurities by washing with solvent. Finally, the isolated protein is detached from the column using acids or other auxiliary reagents. However, this process has a disadvantage: the purified target protein can be damaged in particular during the last step.
A team led by Arne Skerra, Professor of Biological Chemistry at TUM, has therefore developed a new approach: “We use a physical mechanism instead of chemical reagents. Our technology is fundamentally different from the conventional method, being both more gentle and more efficient,” says Arne Skerra.
The “Azo-tag”: a molecular appendix serves as an anchor
The new method also uses a chromatography column filled with a porous carrier material. However, the difference is that LED lights are placed around the column and, furthermore, a small molecular appendix is attached to the target protein.
This minimalistic accessory, dubbed Azo-Tag, was developed by Peter Mayrhofer, Markus Anneser and Stefan Achatz together with Arne Skerra at the Chair of Biological Chemistry on the basis of the light-sensitive chemical group “azo-benzene”. The Azo-tag can change its shape under light exposure and serves as a molecular anchor for the target protein: in daylight or in the dark, the target protein binds specifically to the carrier material in the chromatography column via this anchor. Any other contaminating substances and impurities can be washed out, whereas the target protein with its anchor is retained.
However, if the LED lights are then switched on and the column is irradiated with mild UV light with a wavelength of 355 nanometers, the tag changes its shape. Simply speaking, it is repelled from the carrier material so that the target protein with its Azo-tag is washed out of the column in a pure, concentrated and undamaged form. Isolated in this manner, the protein can be used directly for further studies — without additional purification steps.
More efficient than conventional chromatography and potential for further development
The Chair of Biological Chemistry is now regularly using this method and has already been able to purify antibodies against breast cancer. Currently, a small version of the apparatus is being used in the laboratory. The chromatography column measures less than one centimeter in diameter, but the team expects it could also be built on a larger scale.
There are also further plans, says Arne Skerra, who has filed with his coworkers a patent for this novel method: “We are currently working on automating the processes to make them even more efficient, especially for high-throughput drug development in pharmaceutical or biotechnology companies.”