Application of a green fluorescent fusion protein to study protein-protein interactions by electrophoretic methods.
A screening procedure for protein-protein interactions in cellular extracts using a green fluorescent protein (GFP) and affinity capillary electrophoresis (ACE) was established. GFP was fused as a fluorescent indicator to the C-terminus of a cyclophilin (rDmCyp20) from Drosophila melanogaster. Cyclophilins (Cyps) belong to the ubiquitously distributed enzyme family of peptidyl-prolyl cis/trans isomerases (PPlases) and are well known as cellular targets of the immunosuppressive drug cyclosporin A (CsA). The PPlase activity of the GFP fused rDmCyp20 as well as the high affinity to CsA remain intact. Using native gel electrophoresis and ACE mobility-shift assays, it was demonstrated that the known moderate affinity of Cyp20 to the capsid protein p24 of HIV-1 was detectable in the case of rDmCyp20 fused to the fluorescent tag. For the p24 / rDmCyp20-GFP binding an ACE method was established which allowed to determine a dissociation constant of Kd = 20+/-1.5 x 10(-6) M. This result was verified by size-exclusion chromatography and is in good agreement with published data for the nonfused protein. Moreover the fusion protein was utilized to screen rDmCyp20-protein interactions by capillary electrophoresis in biological matrices. A putative ligand of rDmCyp20 in crude extracts of embryonic D. melanogaster was discovered by mobility-shift assays using native gel electrophoresis with fluorescence imaging and ACE with laser-induced fluorescence detection. The approach seems applicable to a wide range of proteins and offers new opportunities to screen for moderate protein-protein interactions in biological samples.
Kiessig
S
S
Max Planck Research Division Enzymology of Protein Folding, Halle/Saale, Germany.
Reissmann
J
J
Rascher
C
C
Küllertz
G
G
Fischer
A
A
Thunecke
F
F
D019075:Electrophoresis, Capillary / Q000379:methods*; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins; D011485:Protein Binding; D011506:Proteins / Q000032:analysis*
D008164:Luminescent Proteins; D011506:Proteins; D049452:Green Fluorescent Proteins