Lights, column, action: a cleaner method for protein purification
Researchers from TUM report a novel method of protein purification, which is light controlled one-step column ‘Excitography’.
Scientists at the Technical University of Munich (TUM; Germany) have developed a UV light-based protein purification method that is gentler and more efficient than traditional techniques. By eliminating the need for harsh chemicals this method preserves protein integrity unlike previous procedures, offering exciting and widespread applications within pharmaceutical research.
Protein purification is a key technique in many scientific disciplines across industry and academia. For decades affinity chromatography has been the go-to method for purifying a protein of interest. However, the chemical conditions required to immobilize a protein-of-interest (POI) can be severe and often run the risk of contamination, or jeopardize the functionality of the protein due to conformational changes occurring during capture or elution. Often, after affinity chromatography proteins will require additional purification steps, which can also damage the target protein.
Arne Skerra (a Professor of Biological Chemistry at TUM) and colleagues have developed a novel method for protein purification that doesn’t require reagents or extreme pH.
“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,” explains Skerra.
In essence, Skerra and colleagues describe a protein purification method that is controlled by UV light. They have developed the Azo-tag, a non-canonical amino acid that is fused to the protein of interest prior to purification. The Azo-tag is based on the organic molecule azobenzene that has some unique physical properties. Azobenzene is an example of a molecule known as a diastereomer. Diastereomers are molecules that are identical in composition but have two absolute conformations, in this instance cis and trans. Azobenzene is comprised of two benzene rings, connected via a central nitrogen–nitrogen double bond. When the benzene rings lie on the same side of the double bond, this is cis-azobenzene, when the benzene lings lie on opposite sides of the bond, the molecule is known as trans-asobenzene.
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The team use a specially designed chromatography matrix that is selective to the trans-isomer of the Azo-tag. When The trans-Azo-tag is exposed to UV light at a wavelength of 355nm, it enters into an electronically excited state, causing it to undergo a physical change to its cis-isomer. This causes the tagged proteins to be eluted from the column in their pure, undamaged and concentrated forms. Other unwanted and untagged molecules can simply be washed out of the column. Isolating the protein in this form means it is ready for further studies without requiring extra purification steps.
This method has now become de rigueur protocol in the Skerra laboratory and already has several applications, including antibody purification. Although for the moment this technique has only been realized on a small scale in the laboratory, the team hope that it can one day be utilized in an industrial capacity.
Commenting on the future of the technique, Skerra adds “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”.
