Affiliation:
1. grid.411818.5 0000 0004 0498 8255 Department of Biosciences Jamia Millia Islamia 110025 New Delhi India
2. grid.411818.5 0000 0004 0498 8255 Department of Chemistry Jamia Millia Islamia 110025 New Delhi India
Abstract
Abstract
Due to the potential toxicity of mercury, there is an immediate need to understand its uptake, transport and flux within living cells. Conventional techniques used to analyze Hg2+ are invasive, involve high cost and are less sensitive. In the present study, a highly efficient genetically encoded mercury FRET sensor (MerFS) was developed to measure the cellular dynamics of Hg2+ at trace level in real time. To construct MerFS, the periplasmic mercury-binding protein MerP was sandwiched between enhanced cyan fluorescent protein (ECFP) and venus. MerFS is pH stable, offers a measurable fluorescent signal and binds to Hg2+ with high sensitivity and selectivity. Mutant MerFS-51 binds with an apparent affinity (K d) of 5.09 × 10−7 M, thus providing a detection range for Hg2+ quantification between 0.210 µM and 1.196 µM. Furthermore, MerFS-51 was targeted to Escherichia coli (E. coli), yeast and human embryonic kidney (HEK)-293T cells that allowed dynamic measurement of intracellular Hg2+ concentration with a highly responsive saturation curve, proving its potential application in cellular systems.
Funder
Department of Biotechnology, Govt of India
University Grants Commission, Govt of India
Publisher
Oxford University Press (OUP)
Subject
Applied Microbiology and Biotechnology,Biotechnology,Bioengineering
Cited by
8 articles.
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