Directed Enzyme and Pathway Evolution

Vick, Jacob E.; Schmidt‐Dannert, Claudia

doi:10.1002/9780470627303.ch2

Cited by 1 publication

(1 citation statement)

References 161 publications

(171 reference statements)

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The utilization of proteins for bioprocessing, sensing, and medical treatment is widespread over a number of industries including agricultural, textile, pharmaceutical, energy, and chemical processing 3–5. Proteins have a number of advantages over nonbiological chemicals and chemical processes due to: (1) superior chemo‐, regio‐, and stereospecificity of enzymes3, 6–8; (2) green sustainability (renewable, biodegradable, nontoxic, milder reaction conditions, less energy consumption)3, 5, 9–11; and (3) optimization through protein engineering 12, 13. However, difficulties in recovery, reuse, and long‐term stability limit the far‐reaching industrial potential of proteins 14.…”

Section: Introductionmentioning

confidence: 99%

Enhanced protein stability through minimally invasive, direct, covalent, and site‐specific immobilization

Smith¹,

Wu²,

Varner³

et al. 2013

Biotechnology Progress

View full text Add to dashboard Cite

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.

show abstract