Site-specific chemical protein conjugation using genetically encoded aldehyde tags

被引：185

作者：

Rabuka, David ^{[2
]}

Rush, Jason S. ^{[1
]}

deHart, Gregory W. ^{[2
]}

Wu, Peng ^{[1
]}

Bertozzi, Carolyn R. ^{[1
,3
,4
]}

机构：

[1] Univ Calif Berkeley, Dept Chem, Berkeley, CA 94720 USA

[2] Redwood Biosci Inc, Emeryville, CA USA

[3] Univ Calif Berkeley, Dept Mol & Cell Biol, Berkeley, CA 94720 USA

[4] Univ Calif Berkeley, Howard Hughes Med Inst, Berkeley, CA 94720 USA

来源：

NATURE PROTOCOLS | 2012年 / 7卷 / 06期

基金：

美国国家卫生研究院;

关键词：

FORMYLGLYCINE-GENERATING ENZYME; SULFATASES;

D O I：

10.1038/nprot.2012.045

中图分类号：

Q5 [生物化学];

学科分类号：

071010 ; 081704 ;

摘要：

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 alpha-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.

引用

收藏

页码：1052 / 1067

页数：16

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