Site-specific chemical protein conjugation using genetically encoded aldehyde tags

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

doi:10.1038/nprot.2012.045

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 journal › Article › peer-review

206 Scopus citations

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 language	English (US)
Pages (from-to)	1052-1067
Number of pages	16
Journal	Nature Protocols
Volume	7
Issue number	6
DOIs	https://doi.org/10.1038/nprot.2012.045
State	Published - Jun 2012
Externally published	Yes

ASJC Scopus subject areas

General Biochemistry, Genetics and Molecular Biology

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10.1038/nprot.2012.045

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title = "Site-specific chemical protein conjugation using genetically encoded aldehyde tags",

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.",

author = "David Rabuka and Rush, {Jason S.} and Dehart, {Gregory W.} and Peng Wu and Bertozzi, {Carolyn R.}",

note = "Funding Information: acKnoWleDGMents 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.",

year = "2012",

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doi = "10.1038/nprot.2012.045",

language = "English (US)",

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AU - Rabuka, David

AU - Rush, Jason S.

AU - Dehart, Gregory W.

AU - Wu, Peng

AU - Bertozzi, Carolyn R.

N1 - Funding Information: acKnoWleDGMents 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.

PY - 2012/6

Y1 - 2012/6

N2 - 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.

AB - 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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Site-specific chemical protein conjugation using genetically encoded aldehyde tags

Abstract

ASJC Scopus subject areas

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