(546b) Enabling Directed Evolution in Materials Development: High-Throughput Screening of Streptavidin-Binding Proteins in Self-Assembled Solid Films

Proteins have unique binding, enzymatic, and structural abilities due to the environment shaping their function over time via evolution. These desirable functions, which are difficult to replicate synthetically, can be incorporated into protein-based materials, such as biosensors, industrial catalysis, waste treatment, among many other applications and devices. In many of these applications, to maintain desirable performance, the proteins must retain high activity, selectivity, and stability in hostile conditions, which motivates a need to immobilize the protein of interest. However, many of the high-throughput screening techniques used for directed evolution to improve protein performance are not readily conducive to immobilized protein modifications, as would be required in paper-based biosensors.

In this work, a high-throughput platform to assess the binding affinity for immobilized sensing proteins is established. A library of fusion proteins, consisting of an elastin-like polypeptide block (ELP), one of 25 variants of Sso7d obtained via directed evolution (screened for affinity to streptavidin), and a coiled-coil order-directing sequence (ZE), was generated by Gibson Assembly cloning. Of the 25 total fusion proteins, 21 were successfully expressed and were able to be purified in a well-plate format using ELP-based precipitation techniques. Unlike in many directed evolution techniques, using purified products is essential when testing material properties, so the dramatic increase in the throughput of the purification is necessary to build larger libraries to test. These fusion proteins were tested in both dilute solution conditions, via biolayer interferometry, and in self-assembled films with fluorescence microscopy. Some variants showed high affinity in both the solution and film conditions, while others had high affinity in the solution-based assay and low affinity in the film-based assay, likely due to inaccessibility of the binding domains. These differences are indicative of the need to continue to develop tools to translate the efficiencies of directed evolution to protein materials systems.