(548b) Oligomericity Is an Important Indicator in the Kinetic Stability of Biocatalytic Proteins

Oligomeric status of proteins is an important and often undervalued metric to determine the stability of proteins. Unfavorable oligomeric states can lead to aggregation and activity loss in a protein sample, making such a sample less amenable to use in biocatalytic process. In this work, we tested oligomeric state and melting temperature for several amine dehydrogenases and cofactor-regenerating enzymes with OmniSEC and Nanotemper instruments. The experiments were conducted at multiple temperatures and protein concentrations, with and without the presence of a self-assembling glutamate-rich leucine zipper fusion tag. These results were then correlated with the corresponding residual activities to characterize the effects of oligomeric status on protein activity and stability.

The results indicate there is a correlation with a uniform oligomeric presence and kinetic stability. Proteins which are purified with a single oligomer tend to retain higher activity and a more uniform oligomeric state over the course of several days, while those with a broader initial distribution tend to lose activity and aggregate more quickly. The addition of leucine zipper tags dramatically shifts the initial oligomeric states of a given protein in the absence of its binding partner, demonstrating the need to consider the effects of tags on not only the kinetics of an enzyme, but also its quaternary behavior.