Site-specific chemical protein conjugation using genetically encoded aldehyde tags

David Rabuka, Jason S. Rush, Gregory W. Dehart, Peng Wu, Carolyn R. Bertozzi

Research output: Contribution to journalArticle

141 Citations (Scopus)

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.

Original languageEnglish (US)
Pages (from-to)1052-1067
Number of pages16
JournalNature Protocols
Volume7
Issue number6
DOIs
StatePublished - Jun 2012
Externally publishedYes

Fingerprint

Aldehydes
Proteins
Cysteine
Bearings (structural)
Enzymes
Nucleophiles
Consensus Sequence
Recombinant Proteins
Purification
Cells
N-formylglycine
Cell Line

ASJC Scopus subject areas

  • Biochemistry, Genetics and Molecular Biology(all)

Cite this

Rabuka, D., Rush, J. S., Dehart, G. W., Wu, P., & Bertozzi, C. R. (2012). Site-specific chemical protein conjugation using genetically encoded aldehyde tags. Nature Protocols, 7(6), 1052-1067. https://doi.org/10.1038/nprot.2012.045

Site-specific chemical protein conjugation using genetically encoded aldehyde tags. / Rabuka, David; Rush, Jason S.; Dehart, Gregory W.; Wu, Peng; Bertozzi, Carolyn R.

In: Nature Protocols, Vol. 7, No. 6, 06.2012, p. 1052-1067.

Research output: Contribution to journalArticle

Rabuka, D, Rush, JS, Dehart, GW, Wu, P & Bertozzi, CR 2012, 'Site-specific chemical protein conjugation using genetically encoded aldehyde tags', Nature Protocols, vol. 7, no. 6, pp. 1052-1067. https://doi.org/10.1038/nprot.2012.045
Rabuka, David ; Rush, Jason S. ; Dehart, Gregory W. ; Wu, Peng ; Bertozzi, Carolyn R. / Site-specific chemical protein conjugation using genetically encoded aldehyde tags. In: Nature Protocols. 2012 ; Vol. 7, No. 6. pp. 1052-1067.
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