A fluorophore ligase for site-specific protein labeling inside living cells

被引：222

作者：

Uttamapinant, Chayasith ^{[1
]}

White, Katharine A. ^{[1
]}

Baruah, Hemanta ^{[1
]}

Thompson, Samuel ^{[1
]}

Fernandez-Suarez, Marta ^{[1
]}

Puthenveetil, Sujiet ^{[1
]}

Ting, Alice Y. ^{[1
]}

机构：

[1] MIT, Dept Chem, Cambridge, MA 02139 USA

来源：

PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA | 2010年 / 107卷 / 24期

基金：

美国国家卫生研究院;

关键词：

fluorescence microscopy; biotechnology; enzyme engineering; GREEN FLUORESCENT PROTEIN; ESCHERICHIA-COLI; NUCLEAR ACTIN; LIPOIC ACID; CRYSTAL-STRUCTURE; MAMMALIAN-CELLS; FUSION PROTEIN; IN-VIVO; PROBES; TAG;

D O I：

10.1073/pnas.0914067107

中图分类号：

O [数理科学和化学]; P [天文学、地球科学]; Q [生物科学]; N [自然科学总论];

学科分类号：

07 ; 0710 ; 09 ;

摘要：

Biological microscopy would benefit from smaller alternatives to green fluorescent protein for imaging specific proteins in living cells. Here we introduce PRIME (PRobe Incorporation Mediated by Enzymes), a method for fluorescent labeling of peptide-fused recombinant proteins in living cells with high specificity. PRIME uses an engineered fluorophore ligase, which is derived from the natural Escherichia coli enzyme lipoic acid ligase (LplA). Through structure-guided mutagenesis, we created a mutant ligase capable of recognizing a 7-hydroxycoumarin substrate and catalyzing its covalent conjugation to a transposable 13-amino acid peptide called LAP (LplA Acceptor Peptide). We showed that this fluorophore ligation occurs in cells in 10 min and that it is highly specific for LAP fusion proteins over all endogenous mammalian proteins. By genetically targeting the PRIME ligase to specific subcellular compartments, we were able to selectively label spatially distinct subsets of proteins, such as the surface pool of neurexin and the nuclear pool of actin.

引用

页码：10914 / 10919

页数：6

共 40 条

[1] New biarsenical Ligands and tetracysteine motifs for protein labeling in vitro and in vivo: Synthesis and biological applications [J].