title: Construction of fluorescence resonance energy transfer vectors and their application in study of structure and function of signal transducers and activators of transcription 1.
abstract: Protein-protein interactions have been studied extensively by green fluorescent protein-based fluorescence resonance energy transfer (FRET). The fluorescent proteins (FP) can be fused either to the N- or C-terminus of a host protein, but it is difficult to predict which order will perturb the host protein the least and provide the largest FRET. Therefore, a researcher needs to fuse host proteins with FP at both the N- and C-termini and test every possible combination (N-N, N-C, or C-C) to promote the energy transfer efficiency. Consequently, researchers required to do many subclonings. Herein, we designed FRET vectors to make them more efficient. The expression vectors of pCFP-YFP and pYFP-CFP were constructed with both cyan fluorescent protein (CFP)-yellow fluorescent protein (YFP) and YFP-CFP coding sequences flanked by two restriction enzyme sites, and with multiple cloning regions in the middle of both coding sequences. To select an optimal combination for FRET detection, we created plasmids encoding various fusion proteins of FP and signal transducers and activators of transcription 1 (STAT1) . We found that the nuclear:cytoplasmic fluorescence intensity ratios of STAT1-FP were significantly higher than those of FP-STAT1 at steady state, and fluorescence redistribution was only observed for STAT1-FP upon interferon gamma (IFNgamma) stimulation. In addition, positive FRET signals were only detected in the C-C interactions of STAT1 homodimer. Taken together, these data indicate that fusing STAT1 at the N-terminus with FP impairs the interactions of unphosphorylated STAT1 homodimers and possibly diminishes its binding with DNA. In contrast, STAT1-FP was functional with respect to its activation. Moreover, the FRET vectors are able to facilitate FRET studies.
authors.affiliation: Guangzhou Medical College, Guangzhou Institute of Respiratory Diseases, Guangzhou 510120, China.
authors.forename: Fujun
authors.initials: F
authors.lastname: Han
authors.forename: Yongfeng
authors.initials: Y
authors.lastname: Luo
authors.forename: Nanhai
authors.initials: N
authors.lastname: Ge
authors.forename: Jun
authors.initials: J
authors.lastname: Xu
mesh_terms: D001483:Base Sequence; D002460:Cell Line; D003470:Culture Media; D017931:DNA Primers; D019281:Dimerization; D031541:Fluorescence Resonance Energy Transfer; D008856:Microscopy, Fluorescence; D010957:Plasmids; D050794:STAT1 Transcription Factor / Q000737:chemistry* / Q000502:physiology*
chemical_list: D003470:Culture Media; D017931:DNA Primers; D050794:STAT1 Transcription Factor