Engineering Visible-Light Responsive Protein-Polymers

Background: The design of light-responsive proteins can transform our ability to control, manipulate, analyze and engineer biological systems. Here, we focus on Elastin Like Polypeptides (ELPs), polymer-like proteins that can undergo a reversible temperature-mediated transition from soluble to insoluble phase. This transition occurs at a particular temperature that depends on the ELP composition. Our hypothesis is that multi-site and site-specific incorporation of visible-light responsive nonstandard amino acids (nsAAs) in the ELP sequence would enable us to control the solubility of these proteins using light, thereby creating photoswitchable protein-based polymers.

Methods: Using a genomic-engineering based directed evolution approach, we developed an orthogonal translation system (OTS) capable of incorporating visible-light responsive nsAAs.
Using these OTSs we generated ELP variants with varying numbers of ‘TAG’ codons for incorporation of the nsAAs at these positions. The photoswitchable behavior of nsAA-ELPs was characterized by examining the change in the ELP transition temperature with light irradiation.

Results: Two different OTSs were found to have efficient activity in the incorporation of two different visible-light responsive nsAAs. ELPs containing varying amounts of these nsAAs were produced, purified and their Tts were determined in exposure to the affecting wavelengths.
Increasing amounts of incorporated nsAA showed increasing Tt differences between the isomers.

Conclusion: We successfully produced recombinant ELP-based protein polymers and showed significant Tt changes using visible-light irradiation, and thus created a photoswitch able to control the solubility of these proteins. Such ELPs can be used to control the solubility of fusion proteins, to form light-responsive nanostructures, and as model, photoswitchable, intrinsically disordered proteins.