Beyond protein tagging: Rewiring the genetic code of fluorescent proteins - A review.

Fluorescent proteins (FP) are an integral part of modern biology due to its diverse biochemical and photophysical properties. The boundaries of FP have been extended through conventional mutagenesis and directed evolution approaches. Engineering of FP based on the standard genetic code consisting of 20 amino acids with limited functional groups restrict its diversification. Degeneracy of genetic code has helped in covering this substantial gap through genetic code engineering, wherein introduction of unnatural amino acid (UAA) analogues resulted in a collection of FP with varying properties. This review features the work carried till date in the area of FP incorporated with UAAs and explores strategies employed for incorporation, impact of UAAs in chromophore and surrounding residues and changes in inherent properties of FP. The long-standing association of FP as a tool for high throughput screening of orthogonal aaRS/tRNA pairs used in site specific incorporation of UAAs is expounded. Insertion of UAAs in FP has enabled their use in contemporary fields such as biophotovoltaics, bioremediation, biosensors, biomaterials and imaging of acidic vesicles. Thus, expansion of genetic code of FP is envisaged to rejig the existing spectra of colors and future research initiative in this direction is expected to glow brighter and brighter.