Monitoring of in vitro and in vivo translation of green fluorescent protein and its fusion proteins by fluorescence correlation spectroscopy.
BACKGROUND
Because the process of protein translation is an event of sparse molecules, the measurement requires high sensitivity. One of the candidates for studying the molecules is fluorescence correlation spectroscopy (FCS), which gleans quantitative information from fluctuating fluorescence signals in a diluted solution.


METHODS
Using FCS, the translation products of expression plasmid for green fluorescent protein (GFP) and its fusion proteins were measured in vitro and in vivo.


RESULTS
In in vitro translation, the number of products increased linearly for 90 min upon concentration of the plasmid. The autocorrelation function for GFP was fitted with a one-component model with a diffusion time of 0.18 ms, which was identical to the value expected from the molecular weight. In the cases of GFP- tagged hypoxia-inducible factor-1 alpha and glucocorticoid receptor, each fitting result was significantly improved with a two-component model. The slow component with a diffusion time of 6 ms appeared to be related to the ribosome or polysome. In response to the addition of dexamethasone, the nuclear translocation from cytosol clearly induced the decrease in number of molecules in the focal point.


CONCLUSIONS
FCS permits monitoring of the number of molecules translated in vitro and in vivo, the translation rate, and the molecular weight.
Nomura
Y
Y
Laboratory of Supramolecular Biophysics, Research Institute for Electronic Science, Hokkaido University, Sapporo, Japan. ynomura@imd.es.hokdai.ac.jp
Tanaka
H
H
Poellinger
L
L
Higashino
F
F
Kinjo
M
M
D000818:Animals; D019556:COS Cells; D002522:Chlorocebus aethiops; D004268:DNA-Binding Proteins / Q000235:genetics*; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins; D019382:Human Growth Hormone / Q000235:genetics*; D006801:Humans; D051793:Hypoxia-Inducible Factor 1; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D008164:Luminescent Proteins / Q000235:genetics*; D009687:Nuclear Proteins / Q000235:genetics*; D014176:Protein Biosynthesis; D011993:Recombinant Fusion Proteins / Q000235:genetics; D013050:Spectrometry, Fluorescence; D014157:Transcription Factors
D004268:DNA-Binding Proteins; C497442:HIF1A protein, human; D051793:Hypoxia-Inducible Factor 1; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D008164:Luminescent Proteins; D009687:Nuclear Proteins; D011993:Recombinant Fusion Proteins; D014157:Transcription Factors; D019382:Human Growth Hormone; D049452:Green Fluorescent Proteins