Warfighters going into extreme conditions, such as desert heat, soon will have more rugged detectors against biological weapons, thanks to a protein derived from an animal accustomed to living in that environment.
Recent work managed by DTRA CB/JSTO’s Dr. Ilya Elashvili and performed by researchers at the U.S. Naval Research Laboratory (NRL), led by Dr. Ellen Goldman, highlighted two methods to increase the utility of single domain antibodies (sdAbs).
These sdAbs are binding domains derived from the heavy chain antibodies found in camelids such as camels and llamas, by further enhancing their melting temperature and solubility.
A herd of camels passes by a U.S. Marine patrol, in Kajaki Sofla, Nov. 4, 2011. (U.S. Marine Corps photo by Cpl. James Clark/Released)
Although sdAbs are typically more soluble and thermally stable than other antibody-derived binding domains, increasing their ability to survive high heat challenges can improve the performance of field portable detection devices in austere environments.
These studies were reported in a pair of articles: “Enhanced stabilization of a stable single domain antibody for SEB toxin by random mutagenesis and stringent selection” published in Protein Engineering Design and Selection, and “Negative tail fusions can improve ruggedness of single domain antibodies” published in Protein Expression and Purification.
SdAbs, recombinantly-expressed binding domains, offer alternative binding reagents that provide the affinity and specificity similar to traditional antibodies, but they are much more rugged when exposed to harsh chemicals or high temperatures.
Not only do the reagents withstand denaturation better at higher temperatures, but sdAbs also often recover their 3-D structure and binding ability even after denaturation at high temperatures, unlike traditional antibodies which lose their ability to recover.
However, not all sdAbs possess this ability to recover, and some sdAb clones are prone to irreversible aggregation after heat denaturation.
Therefore, understanding how to manipulate sdAbs to resist aggregation can result in improved reagents with better shelf life and reduced logistical demands, such as shipping and storing without refrigeration.
The team reported earlier the development of an A3 sdAb, which binds with excellent affinity to the potential biothreat agent Staphylococcal enterotoxin B (SEB) and possesses one of the highest melting temperatures (Tm of 83.5°C) yet reported (see the December 2013 JSTO in the News article, “Unraveling the Secrets of Protein Stability”). In a demonstration to push its melting temperature up closer to boiling, the A3 sdAb underwent a process termed random mutagenesis and stringent selection.
A library of variants was constructed in which amino acid changes were introduced randomly through the protein.
The variants were heated and those that retained their ability to bind antigen were selected. Through this process, a derivative of A3 was isolated that had a melting temperature 6.5-degree Celsius higher, while maintaining its ability to recognize toxin with high affinity. This demonstrated that even an sdAb with an extraordinarily high melting temperature is able to be improved.
This strategy should also be applicable to sdAbs with more typical melting temperatures.
Engineering the ability of sdAbs to resist aggregation is another method to improve these reagents. The scientists found that improvement could be accomplished by appending a negatively charged segment to sdAbs.
The sdAbs with the negative tail were much less prone to aggregate when kept for extended periods of time at high concentration above their melting temperature. In some cases, this did not translate into improved ability to function after heating, but in others, such as with the anti-SEB sdAb A3, the fusion of the negatively charged segment led to both aggregation resistance and improved binding performance after heating.
An unexpected result of this work was that sdAb proteins with the negative segment were able to refold after heat denaturation into an active state even when produced under circumstances that typically abolish refolding ability.
This work demonstrates that sdAbs can be engineered to increase their melting temperatures and improve the probability of function after heating above their melting temperature at high concentrations for long periods of time.
The methods used to improve the sdAb should be generalizable and could be applied to any sdAb reagent. These are important advancements towards the realization of sdAbs as improved binding reagents for integration into antibody based biosensors.
Story from the Defense Threat Reduction Agency
Chemical and Biological Technologies Department
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