Fusion proteins of a truncated mutant of myosin subfragment-1 (S1dC) and green fluorescent protein (GFP) wereexpressed 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 lowbackground 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 formand/or in large quantities in conventional heterologous expressionsystems.
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