Site-Specific Dual Labeling of Proteins by Using Small Orthogonal Tags at Neutral pH

被引：8

作者：

Gruenewald, Jan ^{[1
]}

Jones, David H. ^{[1
]}

Brock, Ansgar ^{[1
]}

Chiu, Hsien-Po ^{[1
]}

Bursulaya, Badry ^{[1
]}

Ng, Kenneth ^{[1
]}

Vo, Todd ^{[1
]}

Patterson, Paula ^{[1
]}

Uno, Tetsuo ^{[1
]}

Hunt, James ^{[2
]}

Spraggon, Glen ^{[1
]}

Geierstanger, Bernhard H. ^{[1
]}

机构：

[1] Novartis Res Fdn, Genom Inst, San Diego, CA 92121 USA

[2] Novartis Inst Biomed Res, Horsham RH12 5AB, W Sussex, England

来源：

CHEMBIOCHEM | 2014年 / 15卷 / 12期

关键词：

biosensors; FRET; IgE; phosphopantetheinyl transferase; pyrroline-carboxy-lysine; RESONANCE ENERGY-TRANSFER; PYRROLINE-CARBOXY-LYSINE; UNNATURAL AMINO-ACIDS; CONFORMATIONAL-CHANGES; FRET; IGE; BIOSENSORS; DYNAMICS; CELLS; SFP;

D O I：

10.1002/cbic.201402204

中图分类号：

Q5 [生物化学]; Q7 [分子生物学];

学科分类号：

071010 ; 081704 ;

摘要：

To expand the utility of proteinaceous FRET biosensors, we have developed a dual-labeling approach based on two small bio-orthogonal tags: pyrroline-carboxy-lysine (Pcl) and the S6 peptide. The lack of cross-reactivity between those tags enables site-specific two-color protein conjugation in a one-pot reaction. Moreover, Pcl/S6 dual-tagged proteins can be produced in both bacterial and mammalian expression systems, as demonstrated for Z domain and IgE-Fc, respectively. Both proteins could be efficiently dual-labeled with FRET-compatible fluorescent dyes at neutral pH. In the case of IgE-Fc, the resulting conjugate enabled the monitoring of IgE binding to its high-affinity receptor Fc epsilon RI, which is a key event in allergic disease.

引用

页码：1787 / 1791

页数：5

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