(188bw) Methods for High Throughput Fabrication and Screening of Protein-Based Materials

Enzymes are attractive catalysts for a variety of applications, including pharmaceutical synthesis, biosensors, and commodity chemical synthesis. They present several advantages over traditional catalysts due to their high specificity, stereoselectivity, and ability to operate at mild conditions in aqueous environments. Additionally, efforts in protein engineering have greatly expanded enzyme functionality beyond naturally occurring reactions. However, fabrication of enzymes into biofunctional devices requires incorporation into a material, and it is well-known that local environment plays an important role in protein function. It follows that engineering of functional protein materials must not only consider the solution-phase reactivity of proteins, but also the local environment of the protein in its end-use state. The goal of this work is to develop a procedure for high throughput fabrication and screening of protein-based materials that allows for selection of a desired enzymatic functionality in the end-use immobilized state.

Here, we demonstrate the success of this high throughput approach using several model globular proteins. The high-throughput fabrication approach is centered around fusion of an elastin-like polypeptide (ELP) tag to the functional protein of interest. This ELP tag serves two purposes. First, the Chilkoti group has shown that fusion of an ELP tag to a globular protein allows for purification of the target protein via inverse transition cycling (ITC). Here, we demonstrate the success of this cycling approach on ELP fusions in a well-plate format, allowing for purification of an entire protein library without the need for expensive chromatography-based techniques. The second purpose of this ELP tag is in the actual material formation. Work in the Olsen lab has demonstrated that fusion of an ELP tag to globular proteins leads to solid-state nanostructure formation similar to that observed in protein-polymer bioconjugates, and that the function of the globular protein is retained in these solid state materials. We find that this behavior holds for the ELP fusions purified in a well-plate format, and that solid-state films that retain globular protein function can be formed using this strategy. Finally, protein films cast in this fashion can be permanently immobilized at the bottom of a well-plate using glutaraldehyde crosslinking chemistry.