Single molecular assay of individual ATP turnover by a myosin-GFP fusion protein expressed in vitro.
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.
Iwane
A H
AH
Department of Biophysical Engineering, Faculty of Engineering Science, Osaka University, Toyonaka, Japan. iwane@bpe.es.osaka-u.ac.jp
Funatsu
T
T
Harada
Y
Y
Tokunaga
M
M
Ohara
O
O
Morimoto
S
S
Yanagida
T
T
D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000378:metabolism*; D000818:Animals; D002645:Chickens; D005456:Fluorescent Dyes / Q000378:metabolism; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D008856:Microscopy, Fluorescence / Q000379:methods; D015879:Myosin Subfragments / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism
D005456:Fluorescent Dyes; D008164:Luminescent Proteins; D015879:Myosin Subfragments; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D000255:Adenosine Triphosphate