Single molecular assay of individual ATP turnover by a myosin-GFP fusion protein expressed in vitro

FEBS Letters
A H IwaneT Yanagida

Abstract

Fusion proteins of a truncated mutant of myosin subfragment-1 (S1dC) and green fluorescent protein (GFP) were expressed in vitro by T7 RNA polymerase and rabbit reticulocyte lysate. Single S1dC-GFP fusion proteins were clearly seen and their individual ATP turnovers were directly monitored using low background total internal reflection fluorescence microscopy (LBTIRFM), recently developed by our laboratory. LBTIRFM using GFP as a fluorescent tag allowed us to assay functions of single protein molecules expressed in vitro. Thus, the results suggested that this method may be particularly useful to analyze functions of proteins that cannot be produced in an active form and/or in large quantities in conventional heterologous expression systems.

References

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Citations

Feb 4, 1999·Biophysical Journal·Y HaradaT Yanagida
Jul 7, 2001·Trends in Biochemical Sciences·A Ishijima, T Yanagida
Aug 3, 2002·Microbiology and Immunology·Takashi FunatsuHideo Namiki
Feb 28, 2006·Biophysical Journal·Mitsuhiro IwakiToshio Yanagida
Sep 15, 2005·Journal of Biotechnology·Anna Stanisławska-SachadynJózef Kur
Feb 18, 2017·Chemical Reviews·Matthew B StoneSarah L Veatch
Jul 12, 2002·The Journal of Biological Chemistry·Diana ChowDonald A Winkelmann
Dec 26, 2001·Chemical Reviews·W P AmbroseR A Keller

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