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Application of a green fluorescent fusion protein to study protein‐protein interactions by electrophoretic methods
Author(s) -
Kiessig Steffen,
Reissmann Jana,
Rascher Christine,
Küllertz Gerhard,
Fischer Andreas,
Thunecke Frank
Publication year - 2001
Publication title -
electrophoresis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.666
H-Index - 158
eISSN - 1522-2683
pISSN - 0173-0835
DOI - 10.1002/1522-2683(200105)22:7<1428::aid-elps1428>3.0.co;2-z
Subject(s) - green fluorescent protein , capillary electrophoresis , fusion protein , cyclophilin , fluorescence , affinity electrophoresis , chemistry , tandem affinity purification , protein–protein interaction , biochemistry , gel electrophoresis , electrophoretic mobility shift assay , dissociation constant , affinity chromatography , microbiology and biotechnology , biology , enzyme , chromatography , recombinant dna , physics , gene expression , receptor , quantum mechanics , gene
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 (PPIases) and are well known as cellular targets of the immunosuppressive drug cyclosporin A (CsA). The PPIase 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 K d = 20 ± 1.5×10 −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.