Functional Screening Assays
Membrane proteins not only can be difficult to express but also to purify and assay. Many researchers approach the study of membrane proteins by over-expressing recombinant fragments of membrane-associated enzymes and studying their activity in solution. However, this can be a daunting challenge for many assay systems, according to Scott Gridley, Ph.D., director of bioproducts at BlueSky Biotech.
“Membrane-associated proteins derive significant structural, topological, and relational organization from being constrained on the fluid two-dimensional surface of the membrane.”
The company has developed a technology called TDA 2.0™ that more closely replicates native protein structure to enhance functional assays, Dr. Gridley says. “Template-directed assembly, the process of organizing recombinant protein on a membrane surface, such as a liposome, restores biological context resulting in a more relevant set of data. TDA 2.0 is the only commercially available technology that readily replicates the membrane context in a soluble, fluid, chemically defined system compatible with high-throughput screening.”
How does it work? According to Dr. Gridley, “to reproduce membrane association, the enzymes are polyhistidine tagged, and the lipid nanospheres are derivatized with nickel-nitrilotriacetic acid so that polyhistidine tagged proteins bind with high-affinity. Membrane proteins in the context of TDA 2.0 naturally form biologically relevant multimers, and interact with untagged interacting partners to form higher-order complexes without any special effort. We have some purified proteins to replicate pathway interactions in the context of TDA 2.0 in a chemically defined system already available.”
The company currently offers kits for the insulin receptor, insulin-like growth factor receptor tyrosine kinases, and lyn kinase. For the future, Dr. Gridley indicates the company is “developing kits for nearly every receptor tyrosine kinase family member (~70), as well as extending the technology for use with other membrane protein classes.”
While daunting challenges remain in the arena of difficult-to-express proteins, new paradigms in expression science are helping pave the way for solving those problems.