1. Select the fluorescent protein fragments to be used
Several combinations of fluorescent protein fragments support bimolecular fluorescence complementation 11
; those recommended for BiFC analysis are listed in . For most purposes, fragments of YFP truncated at residue 155 (designated YN155 and YC155) are recommended, as they exhibit a relatively high complementation efficiency when fused to many interaction partners, yet produce low fluorescence when fused to proteins that do not interact with each other 2
. Fragments of YFP truncated at residue 173 (designated YN173 and YC173) can also be used 11
, and may exhibit a different efficiency of complementation due to differences in the steric constraints imposed by tethering of the fragments to the protein complex. Fragments of Venus (a mutated GFP with high fluorescence intensity) 22
truncated at either residue 155 or 173 (designated VN155 and VC155 or VN173 and VC173 respectively) produce a significantly brighter fluorescent signal when fused to specific interaction partners 20
. However, these fragments also produce a brighter signal when fused to proteins that do not selectively interact with each other 20
. These fragments have the great advantage that the bimolecular fluorescent complex is readily detectable at 37°C, which avoids the incubation at 30°C that is generally necessary to detect complementation using YFP fragments. Other combinations of fluorescent protein fragments can also be used, especially when using BiFC analysis for the visualization of multiple protein complexes in the same cell 11
2. Determine the sites where the fluorescent protein fragments can be fused to the putative interaction partners
Determine the positions of the fusions empirically to fulfill three criteria.
- First, ensure that the fusions allow the fragments of the fluorescent proteins to associate with each other if the putative partners interact. Information about the structure and location of the interaction interface may be useful to determine optimal positions for the fusions. However, this information is not essential since fusions that can be used for BiFC analysis can be identified by screening multiple combinations of fusion proteins for fluorescence complementation. One strategy for the identification of fusion proteins that allow bimolecular fluorescence complementation is to fuse each of the fluorescent protein fragments to the N- and C-terminal end of each interaction partner, and to test for complementation in all eight combinations that contain both fragments of the fluorescent protein ().
Combinations of fusion proteins to be tested for bimolecular fluorescence complementation
- Second, confirm that fusions do not affect the localization or the stabilities of the proteins by comparing the localization and expression levels of the fusion proteins with those of wildtype proteins lacking the fusions; indirect immunofluorescence and immunoblot analyses can be used.
- Third, test the fusion proteins for all known functions of the endogenous proteins to ensure that the fusions do not affect the functions of the proteins under investigation.
3. Select linkers to connect the fragments to the proteins of interest
The linkers must provide flexibility for independent motion of the fluorescent protein fragments and the interaction partners, allowing the fragments to associate when the proteins interact. We have used the RSIAT and RPACKIPNDLKQKVMNH linker sequences in many fusion constructs used for BiFC analysis 2, 11
. These linkers have been used for the visualization of interactions between many structurally unrelated proteins. The sequence AAANSSIDLISVPVDSR encoded by the multiple cloning sites of the pCMV-FLAG vector (Sigma) has also been successfully used as a linker in many BiFC experiments. A peptide sequence designed to be flexible such as (GGGS)n
can also be used, although it can potentially affect the degradation of the fusion protein. Although these linker sequences have worked well for the proteins examined previously, it is possible that linkers of a different length or sequence are optimal for BiFC analysis of interactions between other proteins.
4. Select a cell culture system
Choose a cell culture system that represents the biological context to be investigated, and allows efficient introduction of DNA into a large fraction of the cells. Cells that grow as an adherent monolayer are generally easier to image. The BiFC assay has been used for the analysis of protein interactions in many mammalian cell lines including COS-1, HEK293, HeLa, Hep3B, αTN4, and NIH3T3 cells as well as in intact organisms 2, 10, 12, 18, 19, 23-67
5. Select a strategy for expression of the fusion proteins
Choose either transient expression (A) or stable expression (B) strategies, based on the purpose of the experiment.
A). Transient overexpression of the fusion proteins
This approach may be adequate to determine if a pair of proteins can interact in cells and to determine the subcellular location of the complex. To minimize protein mislocalization and formation of non-native complexes due to overexpression, express the fusion proteins at levels comparable to the endogenous proteins. This can be achieved by using plasmids with weak promoters, by transfecting small amounts of plasmid DNA and by observing the cells as soon as signal is detectable.
B) Expression in stable cell lines
More reproducible levels of expression can be obtained by using inducible expression vectors integrated into the genomes of stable cell lines. This allows for the control of protein expression at relatively uniform levels in the entire cell population and replication of experiments at constant expression levels, independent of transfection efficiency and other factors that are difficult to control in transient assays.
6. Design controls to determine if complementation reflects a specific protein interaction
As fluorescent protein fragments are able to form fluorescent complexes with a low efficiency in the absence of a specific interaction, it is essential to include negative controls in each experiment. Spontaneous complementation is generally reduced when the fragments are fused to proteins that do not interact with each other; appropriate negative controls are fusion proteins in which the interaction interface has been mutated and fused to the fluorescent protein fragments in a manner identical to the wildtype fusion proteins () 2, 10
. Compare the level of expression and localization of the mutated and wild-type fusion proteins by immunoblot and indirect immunofluorescence analyses (using any standard or commercially available method). Quantify and compare the efficiencies of fluorescence complementation between the wild-type and mutated proteins. If there is no prior knowledge of the location or the structural nature of the interaction interface, it is possible to screen for mutations that alter the efficiency of bimolecular fluorescence complementation, and thereby determine if the complementation reflects a specific interaction. The BiFC assay can therefore be used to determine whether two proteins interact in cells without prior knowledge of the location or the structural nature of the interaction interface.
Determination of the specificity of bimolecular fluorescence complementation by mutational analysis
Use some of the numerous fusion proteins whose interactions have been visualized using the BiFC assay as positive controls (see ). However, the failure to detect fluorescence complementation between the proteins under investigation does not demonstrate the absence of an interaction (see ANTICIPATED RESULTS).
7. Prepare plasmids encoding fusion proteins
Construct plasmid expression vectors, using the appropriate vectors, by fusing the sequences encoding the selected fluorescent protein fragments (see ) to the sequences encoding the proteins of interest. Any standard cloning techniques can be used. Whenever possible, test fusions to both the N- and C-terminal ends of the proteins to be investigated (). Construct negative control plasmids that encode mutated non-interacting variants of the proteins (see step 6) using the same strategy. Positive controls should be included to ensure that a known interaction can be detected (see step 6).
8. Prepare cells for transfection
Seed cells the day before transfection at an appropriate density. This density should allow for cell proliferation over the course of the experiment while taking into consideration the effects of cell growth and density on the interaction under investigation. Cluster plates are convenient for processing multiple transfections in parallel. If short-working-distance objectives will be used to visualize the interaction, grow the cells in slide chambers or on glass coverslips.
9. Transfect cells
Transfect cells (using the optimal procedure for the cells) with appropriate amounts (e.g. 0.25 μg) of the BiFC plasmids encoding the fusion proteins when an appropriate confluency (e.g. ~50%) is reached. In parallel, transfect cells with the negative and positive control plasmids. For quantitation of the efficiency of fluorescence complementation, all plasmids should be co-transfected with the same amount of an internal control plasmid (e.g. a plasmid that expresses CFP, see )
10. Allow time for fusion protein expression and fluorophore maturation
Grow cells under conditions appropriate for the cell-type until fluorescence is detected (12 to 36 hr). If necessary, incubate the cells at 30°C with 5% CO2 to promote maturation of the fluorophore and to increase the signal. Results obtained under low temperature conditions should be interpreted with care, as incubation at a lower temperature could alter protein interactions.
11. Remove dead cells
Wash the cells once with an amount of PBS sufficient to remove dead cells and cell debris, then add fresh medium.
12. Observe cells
Image the cells using an inverted fluorescence microscope. When using cells grown on plastic, a long-working-distance objective is convenient, but produces lower signal (due to the lower numerical aperture) than a short-working distance objective. A 20× objective is useful for observing large numbers of cells and can provide general subcellular localization information, whereas 60× or 100× objectives can be necessary for detailed localization within subcellular compartments. For detection of complementation between YFP or Venus fragments an excitation filter with 500 ± 10 nm transmission and an emission filter with 535 ± 15 nm transmission are appropriate. Confirm that fluorescent cells are alive by comparing their morphology to that of non-transfected cells. Cells grown on coverslips can be fixed and individual proteins can be visualized by indirect immunofluorescence analysis. Use protocols for fixation and immunofluorescence that have been established for the cell line and antibody to be used.
13. Establish the levels of protein expression
Compare the levels of fusion protein expression with those of the endogenous proteins by immunoblot analysis. The cultures used for imaging can be subsequently processed for immunoblotting or separate cultures can be prepared in parallel. Use protocols for immunoblotting that have been established to work for the cell line and antibody to be used. Use the ratio between the intensities of the bands corresponding to the transfected and endogenous proteins together with the transfection efficiency to estimate the relative levels of transfected and endogenous proteins. Ideally, the amount of transfected protein should not exceed that of the endogenous proteine in the cells.
14. Analyze the data
Compare the intensities and numbers of fluorescent cells observed when the cells are transfected with the wildtype interaction partners with those observed when the cells are transfected with the negative control constructs. For quantitative analysis of the efficiencies of fluorescence complementation, divide the fluorescence intensities produced by fluorescence complementation by the fluorescence intensities produced by intact fluorescent protein in individual cells (see ). Higher fluorescence intensity and an increased number of fluorescent cells for the wild type proteins is consistent with a specific interaction.