Genetically Encoded Biotin Analogues: Incorporation and Application in Bacterial and Mammalian Cells

Abstract

The biotin–streptavidin interaction is among the strongest known in nature. Herein, the site‐directed incorporation of biotin and 2‐iminobiotin composed of noncanonical amino acids (ncAAs) into proteins is reported. 2‐Iminobiotin lysine was employed for protein purification based on the pH‐dependent dissociation constant to streptavidin. By using the high‐affinity binding of biotin lysine, the bacterial protein RecA could be specifically isolated and its interaction partners analyzed. Furthermore, the biotinylation approach was successfully transferred to mammalian cells. Stringent control over the biotinylation site and the tunable affinity between ncAAs and streptavidin of the different biotin analogues make this approach an attractive tool for protein interaction studies, protein immobilization, and the generation of well‐defined protein–drug conjugates.

Efficient Amber Suppression via Ribosomal Skipping for In Situ Synthesis of Photoconditional Nanobodies

“…Genetic code expansion connects the versatility of chemical synthesis to protein expression in living systems. − Protein modifications can be inserted site-specifically via natural translation machineries . By reprogramming the genetic code, non-canonical amino acids bearing a modification of choice are used in ribosomal polypeptide synthesis. − Non-canonical amino acids have many applications in protein engineering, as they can be equipped with isotopes for structural studies, − photoreactive groups and post-translational modifications for functional studies, − reactive groups for bio-orthogonal coupling, ,,− photocross-linkers, ,,− infrared-active probes to follow conformational dynamics, ,− fluorescent dyes for imaging, − biotin analogues for high-affinity interactions with streptavidin, and stable phosphotyrosine analogues for analysis of signal transduction. , Site-specific incorporation is achieved by suppressing a stop codon with an additional aminoacyl-tRNA-synthetase (aaRS)/tRNA pair. ,,, In bacterial or mammalian cells, the rarest amber codon (TAG) is most often used to minimize suppression throughout the proteome. ,,,, However, the underlying processes of the genetic code expansion are complex and hardly understood. , For efficient protein synthesis, an i...…”

“…11 By reprogramming the genetic code, non-canonical amino acids bearing a modification of choice are used in ribosomal polypeptide synthesis. 12−14 Non-canonical amino acids have many applications in protein engineering, as they can be equipped with isotopes for structural studies, 15−17 photoreactive groups and posttranslational modifications for functional studies, 18−29 reactive groups for bio-orthogonal coupling, 13,15,30−34 photocrosslinkers, 1,22,35−40 infrared-active probes to follow conformational dynamics, 6,36−41 fluorescent dyes for imaging, 42−46 biotin analogues for high-affinity interactions with streptavidin, 47 and stable phosphotyrosine analogues for analysis of signal transduction. 23,48 Site-specific incorporation is achieved by suppressing a stop codon with an additional aminoacyl-tRNA-synthetase (aaRS)/tRNA pair.…”