A study of protein-protein interactions in living cells using luminescence resonance energy transfer (LRET) from Renilla luciferase to Aequorea GFP.
We have previously reported that Escherichia coli and mammalian cells containing a fusion protein consisting of the Renilla luciferase linked to Aequorea GFP exhibited luminescence resonance energy transfer (LRET) from luciferase to GFP in the presence of coelenterazine. In this paper, we describe the construction of two gene fusions in which the cDNA for insulin-like growth factor II (IGF-II) is connected to the cDNA for a "humanized" GFP, and the cDNA for insulin-like growth factor binding protein 6 (IGFBP-6) is linked to a cDNA encoding the Renilla luciferase (RUC). The expression of the fusion gene constructs in CHO cells resulted in single polypeptides with the molecular weights expected for IGF-II-GFP and IGFBP-6-RUC, respectively, based on the use of antibodies against GFP and Renilla luciferase. The secretion of IGF-II-GFP from CHO cells was verified by fluorescence microscopy and the presence of IGFBP-6-RUC in the culture medium was confirmed by luminometry. The interaction between the two known binding partners, IGF-II and IGFBP-6, was monitored by measuring LRET from the IGFBP-6-RUC protein to IGF-II-GFP in the presence of coelenterazine, using a low-light imaging system and spectrofluorometry. Based on these data, luciferase-to-GFP LRET holds great promise for the study of protein-protein interactions in eukaryotic cells in real time.
Wang
Y
Y
Department of Biochemistry, Loma Linda University, CA 92350, USA.
Wang
G
G
O'Kane
D J
DJ
Szalay
A A
AA
D000818:Animals; D001671:Biochemistry / Q000295:instrumentation* / Q000379:methods*; D015153:Blotting, Western; D016466:CHO Cells; D019556:COS Cells; D002130:Calcium Phosphates / Q000378:metabolism; D003001:Cloning, Molecular; D003063:Cnidaria / Q000737:chemistry* / Q000201:enzymology*; D006224:Cricetinae; D018076:DNA, Complementary / Q000378:metabolism; D049452:Green Fluorescent Proteins; D006801:Humans; D007202:Indicators and Reagents / Q000737:chemistry / Q000378:metabolism; D018976:Insulin-Like Growth Factor Binding Protein 6 / Q000378:metabolism; D007335:Insulin-Like Growth Factor II / Q000378:metabolism; D008156:Luciferases / Q000737:chemistry* / Q000378:metabolism; D008164:Luminescent Proteins / Q000737:chemistry* / Q000378:metabolism; D018715:Microscopy, Video; D016254:Mutagenesis, Insertional; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000737:chemistry* / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D014162:Transfection
D002130:Calcium Phosphates; D018076:DNA, Complementary; D007202:Indicators and Reagents; D018976:Insulin-Like Growth Factor Binding Protein 6; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; C485828:alpha-tricalcium phosphate; C485830:tetracalcium phosphate; D049452:Green Fluorescent Proteins; D007335:Insulin-Like Growth Factor II; C485838:calcium phosphate, monobasic, anhydrous; C020243:calcium phosphate; D008156:Luciferases; C485829:calcium phosphate, dibasic, anhydrous