Researchers at the University of Alabama at Birmingham (UAB) have developed a one-step affinity chromatography method for protein purification that they claim is up to 100 times better than existing techniques at achieving high-yield, high-purity, high-activity purification (HHH) of even the most challenging of proteins.
Describing the method in the Proceedings of the National Academy of Sciences, UAB professor of biochemistry and molecular genetics, Dmitry Vassylyev, Ph.D., and colleagues say they used the new system to purify about $400,000-worth of the human transmembrane chaperone protein calnexin within just a few hours. They suggest that the method could have widespread use both for high-throughput research applications and potentially therapeutic protein manufacturing. The UAB researchers describe the method in a paper titled “Efficient, Ultra-High-Affinity Chromatography in a One-Step Purification of Complex Proteins.”
Existing purification methods for complex proteins require multiple steps, can take days to complete, and still achieve much lower yields than the UAB technique, the team claims. “This new method offers a number of crucial advantages to both researchers and the pharmaceutical industry,” Prof. Vassylyev stated. “It is potentially the most efficient and universal tool for high-throughput studies of many significant biological systems and may aid large-scale production of therapeutic proteins.”
The UAB affinity chromatography system, which is simple and can be reused multiple times, exploits the incredibly strong binding affinity of the bacterial toxin colicin E7 DNase (CE7) for its immunity protein, known as immunity protein 7 (Im7). This CE7–Im7 binding affinity is an estimated four to seven times stronger than that of currently used affinity chromatography analogs.
The researchers created an inactive CE7 variant, CL7, which doesn’t display any DNase activity but which does retain full binding affinity for Im7. This CL7 tag is then inserted into the gene for the target protein. The researchers also created an Im7 variant that can be coupled to agarose beads, which are used to fill the chromatography column.
CL7-tagged crude protein lysate is then simply poured into the chromatography column and protein bound to the Im7-bound beads can subsequently be released using an engineered protease site.
The UAB scientists used the system to purify five different complex bacterial and human complex proteins, including calnexin. They claim that their test runs achieved one-step HHH purification, with 97% to 100% purity. They were also able to reuse the chromatography column more than 100 times with no loss of binding capacity.
“The ultra-high-affinity (CL7/Im7) purification system described in this work allows for one-step HHH purification of a wide range of traditionally challenging biological molecules, including eukaryotic, membrane, toxic, and DNA/RNA-binding proteins and complexes,” they conclude.