期刊
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
卷 561, 期 -, 页码 40-44出版社
ACADEMIC PRESS INC ELSEVIER SCIENCE
DOI: 10.1016/j.bbrc.2021.05.013
关键词
Azurin; Lanthanide binding tag; Luminescence quenching; Copper sensing
资金
- National Key Research and Development Program of China [2016YFA0502400, 2018YFA0903300]
- National Natural Science Foundation of China [21878020, U1732264]
- Tianjin Synthetic Biotechnology Innovation Capacity Improvement Project [TSBICIP-KJGG-002-02]
By inserting the lanthanide binding tag (LBT) into the copper binding loop of Azurin protein, a new protein called Az-LBT with two metal bonding centers was created, showing strong luminescence upon coordination with Tb3+ and luminescence quenching upon Cu2+ binding, with high metal specificity and a detection limit of 0.65 μM for Cu2+.
Proteins with hetero-bimetallic metal centers can catalyze important reactions and are challenging to design. Azurin is a mononuclear copper center that has been extensively studied for electron transfer. Here we inserted the lanthanide binding tag (LBT), which binds lanthanide with sub mu M affinity, into the copper binding loop of azurin, while keeping the type 1 copper center unperturbed. The resulting protein, Az-LBT, which has two metal bonding centers, shows strong luminescence upon coordination with Tb3+ and luminescence quenching upon Cu2+ binding. The in vitro luminescence quenching has high metal specificity and a limit-of-detection of 0.65 mu M for Cu2+. With the low background from lanthanide's long luminescence lifetime, bacterial cells expressing Az-LBT in the periplasm also shows sensitivity for metal sensing. (C) 2021 Elsevier Inc. All rights reserved.
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