Proteins are increasingly applied in commercial manufacturing, medicine, and research. Engineering proteins improves many aspects of such applications (e.g., efficiency, safety, quality) through a variety of genetic and chemical techniques. For instance, directed evolution can improve endogenous protein activities, diminish undesired properties, or introduce new activities. Alternatively, bioconjugation to small molecules extends the functionality of proteins beyond what nature can offer. Additionally, protein immobilization in flow enables the recreation or rearrangement of valuable biosynthetic pathways. In my Ph.D. work, I have sought to implement or improve protein engineering techniques to address distinct problems in biotherapeutics, biosensing, and biocatalysis.Proteases exemplify how protein engineering innovates across sectors. For example, proteases in stain removers are engineered to tolerate alkaline conditions, detergents, and extreme temperatures. In research, engineered proteases enable the proteomic mapping of elusive post-translational modifications. We demonstrate the potential of protein engineering in biotherapeutics by retargeting the hyper-specific metalloprotease domain of the FDA-approved botulinum neurotoxin serotype A (LC/A) via directed evolution. The final LC/A variant displayed specificity for a new therapeutic target, infiltrated neurons, and demonstrated reduced toxicity in mice. Importantly, these results guide further engineering of LC/A and related proteases.
Engineered oxidoreductases play an important role in biosensors and basic research. Dihydrofolate reductase (DHFR), for instance, is a model for relating enzymatic conformational motions to biocatalytic events. Many of these studies employed chemically modified DHFR variants, making DHFR attractive for investigating bioconjugation reactions. Indeed, reacting DHFR variants with the maleimide analog pyrocinchonimide revealed higher cysteine-selectivity compared to maleimide. Cystine vs. lysine reactivity is an important factor when constructing protein-drug therapeutics. Moreover, bioconjugating DHFR to boronate enabled the identification of post-translationally modified biomarkers in an electrochemical biosensor. This device sensitively differentiated glycated from non-glycated human serum albumin, an important glycemic control indicator in diabetes.
Engineering reversible immobilization tags into proteins enables their biocatalytic application in a vortex fluidic device. Creating stripes of fluorescent proteins offers a proof of concept for the spatial segregation and rearrangement of high-value biosynthetic pathways. Flowing crude lysate through such devices demonstrates the ability to rapidly capture, purify, and use tagged enzymes for continuous-flow biocatalysis.
