Concerted,
Rapid, Quantitative, and Site-Specific
Dual Labeling of Proteins

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Abstract

Rapid, one-pot, concerted, site-specific labeling of proteins at genetically encoded unnatural amino acids with distinct small molecules at physiological pH, temperature, and pressure is an important challenge. Current approaches require sequential labeling, low pH, and typically days to reach completion, limiting their utility. We report the efficient, genetically encoded incorporation of alkyne- and cyclopropene-containing amino acids at distinct sites in a protein using an optimized orthogonal translation system in E. coli. and quantitative, site-specific, one-pot, concerted protein labeling with fluorophores bearing azide and tetrazine groups, respectively. Protein double labeling in aqueous buffer at physiological pH, temperature, and pressure is quantitative in 30 min.

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Creating new fluorescent probes for cell biology.

Jin. Zhang, Robert E. Campbell, Alice Ting … (2002)

Fluorescent probes are one of the cornerstones of real-time imaging of live cells and a powerful tool for cell biologists. They provide high sensitivity and great versatility while minimally perturbing the cell under investigation. Genetically-encoded reporter constructs that are derived from fluorescent proteins are leading a revolution in the real-time visualization and tracking of various cellular events. Recent advances include the continued development of 'passive' markers for the measurement of biomolecule expression and localization in live cells, and 'active' indicators for monitoring more complex cellular processes such as small-molecule-messenger dynamics, enzyme activation and protein-protein interactions.

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Encoding multiple unnatural amino acids via evolution of a quadruplet-decoding ribosome.

Heinz Neumann, Kaihang Wang, Lloyd Davis … (2010)

The in vivo, genetically programmed incorporation of designer amino acids allows the properties of proteins to be tailored with molecular precision. The Methanococcus jannaschii tyrosyl-transfer-RNA synthetase-tRNA(CUA) (MjTyrRS-tRNA(CUA)) and the Methanosarcina barkeri pyrrolysyl-tRNA synthetase-tRNA(CUA) (MbPylRS-tRNA(CUA)) orthogonal pairs have been evolved to incorporate a range of unnatural amino acids in response to the amber codon in Escherichia coli. However, the potential of synthetic genetic code expansion is generally limited to the low efficiency incorporation of a single type of unnatural amino acid at a time, because every triplet codon in the universal genetic code is used in encoding the synthesis of the proteome. To encode efficiently many distinct unnatural amino acids into proteins we require blank codons and mutually orthogonal aminoacyl-tRNA synthetase-tRNA pairs that recognize unnatural amino acids and decode the new codons. Here we synthetically evolve an orthogonal ribosome (ribo-Q1) that efficiently decodes a series of quadruplet codons and the amber codon, providing several blank codons on an orthogonal messenger RNA, which it specifically translates. By creating mutually orthogonal aminoacyl-tRNA synthetase-tRNA pairs and combining them with ribo-Q1 we direct the incorporation of distinct unnatural amino acids in response to two of the new blank codons on the orthogonal mRNA. Using this code, we genetically direct the formation of a specific, redox-insensitive, nanoscale protein cross-link by the bio-orthogonal cycloaddition of encoded azide- and alkyne-containing amino acids. Because the synthetase-tRNA pairs used have been evolved to incorporate numerous unnatural amino acids, it will be possible to encode more than 200 unnatural amino acid combinations using this approach. As ribo-Q1 independently decodes a series of quadruplet codons, this work provides foundational technologies for the encoded synthesis and synthetic evolution of unnatural polymers in cells.