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Concerted, rapid, quantitative, and site-specific dual labeling of proteins.

Sachdeva A, Wang K, Elliott T, Chin JW - J. Am. Chem. Soc. (2014)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Medical Research Council Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge CB2 0QH, United Kingdom.

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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Incorporating 1 and 2 at positions1and 40 of calmodulin and the kinetics of specific labeling. (a) Expressionwas performed in E. coli bearing ribo-Q1, O-gst-cam1TAG-40AGTA, thePylRS/tRNAUACU pair, and the MjPrpRS/tRNACUA pair. Amino acids 1 and 2 wereused at 2 and 1 mM, respectively. (b) Labeling time course for reactionof CaM11240 with 3 and 4. Each reaction was followed for 2 h byin-gel fluorescence and mobility shift.
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fig2: Incorporating 1 and 2 at positions1and 40 of calmodulin and the kinetics of specific labeling. (a) Expressionwas performed in E. coli bearing ribo-Q1, O-gst-cam1TAG-40AGTA, thePylRS/tRNAUACU pair, and the MjPrpRS/tRNACUA pair. Amino acids 1 and 2 wereused at 2 and 1 mM, respectively. (b) Labeling time course for reactionof CaM11240 with 3 and 4. Each reaction was followed for 2 h byin-gel fluorescence and mobility shift.

Mentions: Next, we investigated labeling 1 and 2 within the same protein. We site-specificallyincorporated 1 and 2 at positions 1 and40 of calmodulin toproduce CaM11240 (Figure 2). We directed the incorporation of amino acid 1 with an MjPrpRS/tRNACUA pairand the incorporation of amino acid 2 with the evolvedPylRS/tRNAUACU pair, which efficiently decodes the quadrupletAGTA codon on orthogonal messages using ribo-Q1.9 Unnatural amino acids were incorporated in response toUAG and AGTA codons at positions 1 and 40 in calmodulin within a GST-calmodulingene on an orthogonal message (O-gst-cam1TAG-40AGTA). Expression of full-length GST-CaM11240 was dependent onthe addition of amino acids 1 and 2 to E. coli, and ESI-MS demonstrated the genetically directedincorporation of amino acids 1 and 2 (Figures 2 and 3, and Supplementary Figure 1). The yield of full length GST-CaM11240 was ∼2 mgper L of culture.


Concerted, rapid, quantitative, and site-specific dual labeling of proteins.

Sachdeva A, Wang K, Elliott T, Chin JW - J. Am. Chem. Soc. (2014)

Incorporating 1 and 2 at positions1and 40 of calmodulin and the kinetics of specific labeling. (a) Expressionwas performed in E. coli bearing ribo-Q1, O-gst-cam1TAG-40AGTA, thePylRS/tRNAUACU pair, and the MjPrpRS/tRNACUA pair. Amino acids 1 and 2 wereused at 2 and 1 mM, respectively. (b) Labeling time course for reactionof CaM11240 with 3 and 4. Each reaction was followed for 2 h byin-gel fluorescence and mobility shift.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4333588&req=5

fig2: Incorporating 1 and 2 at positions1and 40 of calmodulin and the kinetics of specific labeling. (a) Expressionwas performed in E. coli bearing ribo-Q1, O-gst-cam1TAG-40AGTA, thePylRS/tRNAUACU pair, and the MjPrpRS/tRNACUA pair. Amino acids 1 and 2 wereused at 2 and 1 mM, respectively. (b) Labeling time course for reactionof CaM11240 with 3 and 4. Each reaction was followed for 2 h byin-gel fluorescence and mobility shift.
Mentions: Next, we investigated labeling 1 and 2 within the same protein. We site-specificallyincorporated 1 and 2 at positions 1 and40 of calmodulin toproduce CaM11240 (Figure 2). We directed the incorporation of amino acid 1 with an MjPrpRS/tRNACUA pairand the incorporation of amino acid 2 with the evolvedPylRS/tRNAUACU pair, which efficiently decodes the quadrupletAGTA codon on orthogonal messages using ribo-Q1.9 Unnatural amino acids were incorporated in response toUAG and AGTA codons at positions 1 and 40 in calmodulin within a GST-calmodulingene on an orthogonal message (O-gst-cam1TAG-40AGTA). Expression of full-length GST-CaM11240 was dependent onthe addition of amino acids 1 and 2 to E. coli, and ESI-MS demonstrated the genetically directedincorporation of amino acids 1 and 2 (Figures 2 and 3, and Supplementary Figure 1). The yield of full length GST-CaM11240 was ∼2 mgper L of culture.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Medical Research Council Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge CB2 0QH, United Kingdom.

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.

Show MeSH