Video: A gentler and more efficient method to purify proteins with ultraviolet light

Scientists at the Technical University of Munich (TUM), Germany, have developed a method that relies on physics – rather than chemistry – to obtain proteins which are needed for both research and pharmaceuticals manufacturing. 

Using ultraviolet (UV) light they succeeded in purifying proteins from cell extracts with a method which is both more efficient and gentler than previous techniques. Proteins in their pure form are needed by scientists engaged in molecular biology or molecular medicine, for investigation or as active substances. Such proteins can be isolated from natural sources or produced by cells that have been genetically modified.

For more than 50 years affinity chromatography has been the method of choice for protein extraction with the cell extract passing through a chromatography column filled with a porous carrier material. Target proteins bind to the carrier material and are separated from impurities and other chemicals by washing with solvent. 
The isolated protein is then detached from the column by auxiliary reagents or acids, however, during this process the target protein can be damaged.
The method developed at TUM has adapted its column filled with a porous carrier material but the difference is that UV-emitting LED lights are placed around the column and, additionally, a small molecular appendix is attached to the target protein.

“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,” said Professor Arne Skerra, a biological chemist who led the team at TUM.

Dubbed the ‘Azo-Tag’, this technique 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’ changes its shape under exposure to UV light and serves as a molecular anchor for the target protein. In regular daylight (or in the dark) the target protein binds specifically to the carrier material in the chromatography column using this anchor with other contaminants and impurities washing out. But the target protein with its anchor is retained.

When the LED lights are switched on, irradiating the column in UV light – at a 355 nanometres wavelength – the tag changes shape so it is repelled from the carrier material and the target ‘Azo-tagged’ protein being washed out of the column in a pure, concentrated but crucially undamaged form. Isolated in this manner, the protein can be used directly for further studies – without additional purification steps.

Currently, a small version of the apparatus is being used in the laboratory at TUM with the column measuring less than one centimetre in diameter, but the team expects it could also be built on a larger scale.

“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,” added Skerra.

For further reading please visit: 10.1038/s41467-024-55212-y