No abstract

Bioconjugating protein to nonbiological surfaces is an essential component of many promising biotechnologies impacting diverse applications such as medical diagnostics, biocatalysis, biohazard detection, and proteomics. However, to enable the widespread economical use of immobilized-protein technologies, long-term stability, and reusability is essential. To enhance protein stability in harsh conditions, herein we report a minimally invasive and covalent bioconjugation that enables precise control of the immobilization location at potentially any surface-accessible location where the incorporated unnatural amino acid does not impact protein structure and function. Specifically, the PRECISE system is introduced where a uniquely reactive unnatural amino acid was incorporated site-specifically at a prespecified location in GFP using cell-free protein synthesis. The GFP was then directly and covalently attached to superparamagnetic beads by the unnatural amino acid in a single click reaction. The immobilized GFP was probed for retained activity and stability under harsh conditions including freeze-thaw cycling and incubation in urea at elevated temperatures. The immobilized GFP was more stable compared to unattached protein in all cases and for all durations observed. The enhanced stability of the immobilized protein is a promising step towards long-term protein stability for biocatalysis and other immobilized-protein applications.

The incorporation of unnatural amino acids (uAA) can introduce novel functional groups into proteins site-specifically, with important applications in basic sciences and protein engineering. However, uAA incorporation can impact protein expression and functional activity depending on its location within the protein-a process that is not yet completely understood and difficult to predict. Therefore, practical applications often necessitate a time-consuming optimization of uAA location by individual gene cloning, expressions, purification, and evaluations for each location tested. To address this limitation, we introduce a streamlined and versatile in vitro system to rapidly express and screen uAA-containing proteins without cumbersome cell culturing or purification procedures. We utilized this technology to simultaneously screen 24 different t4-lysozyme mutants with different uAA incorporation sites in a matter of hours, compared to weeks-long workflow of conventional methods. Screening data offered a mechanistic explanation to some effects of uAA incorporation on expression and activity. Despite these insights, rational prediction of such effects remained challenging, further confirming the value of a rapid screening approach. Biotechnol. Bioeng. 2017;114: 2412-2417. © 2017 Wiley Periodicals, Inc.

No abstract

No abstract