Abstract
We describe a method for modifying proteins site-specifically using a chemoenzymatic bioconjugation approach. Formylglycine generating enzyme (FGE) recognizes a pentapeptide consensus sequence, CxPxR, and it specifically oxidizes the cysteine in this sequence to an unusual aldehyde-bearing formylglyine. The FGE recognition sequence, or aldehyde tag, can be inserted into heterologous recombinant proteins produced in either prokaryotic or eukaryotic expression systems. The conversion of cysteine to formylglycine is accomplished by co-overexpression of FGE, either transiently or as a stable cell line, and the resulting aldehyde can be selectively reacted with α-nucleophiles to generate a site-selectively modified bioconjugate. This protocol outlines both the generation and the analysis of proteins aldehyde-tagged at their termini and the methods for chemical conjugation to the formylglycine. The process of generating aldehyde-tagged protein followed by chemical conjugation and purification takes 20 d.
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Acknowledgements
D.R. and C.R.B. would like to thank the US National Institutes of Health for financial support (1RC1EB010344-01 and R01GM059907). We also thank S. Zheng and P. Drake for helpful discussions and assistance with the MS protocols.
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All authors discussed all steps of the protocol and applications. D.R. wrote the manuscript and J.S.R., G.W.d., P.W. and C.R.B. revised it.
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The aldehyde-tagging methods are registered patents held by the University of California, Berkeley, and exclusively licensed to Redwood Bioscience.
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Rabuka, D., Rush, J., deHart, G. et al. Site-specific chemical protein conjugation using genetically encoded aldehyde tags. Nat Protoc 7, 1052–1067 (2012). https://doi.org/10.1038/nprot.2012.045
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DOI: https://doi.org/10.1038/nprot.2012.045
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