Abstract
Methods for introducing bioorthogonal functionalities into proteins have become central to protein engineering efforts. Here we describe a method for the site-specific introduction of aldehyde groups into recombinant proteins using the 6-amino-acid consensus sequence recognized by the formylglycine-generating enzyme. This genetically encoded 'aldehyde tag' is no larger than a His6 tag and can be exploited for numerous protein labeling applications.
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Acknowledgements
We thank M. Francis and J. Rush for helpful discussions and D. King and A. Falick for MS expertise. I.S.C. was supported by a postdoctoral fellowship from the US National Institutes of Health. B.L.C. was supported by a predoctoral fellowship from the US National Science Foundation. This work was supported by a grant from the US National Institutes of Health to C.R.B. (GM59907).
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I.S.C. and B.L.C. carried out cloning, expression, purification and fluorescent tagging of the constructs. I.S.C. quantified conversion to fGly and performed multiple epitope assays. B.L.C. performed PEGylation assays. C.R.B. directed the project. All authors worked together to compose the manuscript.
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Supplementary information
Supplementary Fig. 1
Mass spectra confirming the presence of fGly in a tryptic peptide from ald13-Stf0. (PDF 222 kb)
Supplementary Fig. 2
Quantitation of the conversion of cysteine to formylglycine using mass spectrometry. (PDF 156 kb)
Supplementary Fig. 3
Quantitation of the conversion of cysteine to formylglycine using Alexa Fluor 647 C5-aminooxyacetamide labeling. (PDF 251 kb)
Supplementary Fig. 4
PEGylation of ald6-MBP with 2 kDa and 5 kDa aminooxy-PEG. (PDF 184 kb)
Supplementary Table 1
Oligonucleotides used in this study. (PDF 274 kb)
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Carrico, I., Carlson, B. & Bertozzi, C. Introducing genetically encoded aldehydes into proteins. Nat Chem Biol 3, 321–322 (2007). https://doi.org/10.1038/nchembio878
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DOI: https://doi.org/10.1038/nchembio878
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