Much of modern biological research is concerned with the how, when, and where of protein-protein interaction. The need for simpler approaches to study these protein-protein interactions, particularly on a larger scale and especially in intact cells is great. Furthermore, if these interactions could be studied in intact living subjects this would allow additional insights into normal and diseased states. In order to understand such ubiquitous protein interactions, several techniques have been developed and are reviewed elsewhere 1, 2
A protein fragment-assisted complementation (PFAC) strategy for studying protein-protein interaction involves the use of a combination of split reporter gene fragments that encode for split proteins that have relatively low affinity for each other and thus produce low signal. When these same fragments are fused to two interacting proteins of interest, the interaction of the two proteins drives complementation of the split reporters leading to a detectable signal. The PFAC strategy has been developed using a variety of reporter proteins including dihydrofolate reductase3,4
, green fluorescent protein6
, firefly luciferase 7
and renilla luciferase 8
. Several of the currently available techniques for studying protein-protein interactions are restricted to using either cell lysates or intact cells. To extend these applications to small living animals, we and others have adopted a yeast two hybrid system 9, 10
using bioluminescence. To develop a more robust and generalizable strategy for imaging protein interactions in living small animals we previously reported a split firefly luciferase fragment-assisted complementation and intein-mediated reconstitution system11
. The fragment assisted complementation approach was subsequently also studied by others in small animals12
The PFAC strategies based on split bioluminescent reporters (firefly luciferase and renilla luciferase) are particularly useful because of their applicability in imaging protein-protein interactions in intact cells and by direct extension to small living animals. In addition, these bioluminescent reporters hold potential advantages for use in small animals over other reporters particularly due to their low background signal13
. This is in contrast to work with fluorescent reporters (e.g., green fluorescent protein, and red fluorescent protein) and reporters that use fluorescent substrates as a readout (e.g., β-lactamase and β-galactosidase) due to autofluorescence and confounding increases in background signal. We8, 11
and others7, 12, 14
have identified several combinations of split fragments for bioluminescent reporters such as firefly and renilla luciferase that are suitable for studying protein-protein interactions in living animals8, 11, 12, 14
. In addition, we also recently identified several combinations of firefly luciferase enzyme fragments that self-complement without assistance via protein-protein interactions15
. These types of fragments are useful for studying cellular localization of tagged proteins, evaluation of cell macromolecular delivery vehicles, and also for studying cell-cell fusion. We have also recently applied split reporter strategies to study intramolecular folding of the estrogen receptor in intact cells and small living animals16
The sensitivity of the split reporters in studying protein-protein interactions will in general depend on several variables including the affinity of the two interacting proteins of interest. The split firefly luciferase fragments used in our previous study showed a good sensitivity with the interacting protein partners Id/myoD11
, but they failed to produce significant levels of signal with the rapamycin mediated interacting proteins FRB/FKBP12. In the rapamycin mediated interaction strategy the small molecule rapamycin binds to the proteins FRB and FKBP12 and leads to the induction of both homo and heterodimerization between these proteins. This result contrasts with the combination of split firefly luciferase fragments utilized by others that are reported to have a greater level of signal with the rapamycin mediated FRB/FKBP12 interaction system but at a cost of increased background signal12
. The renilla luciferase fragments (Nrluc 229 and Crluc 229) utilized in our previous study show significant levels of protein-protein interaction assisted luciferase signal for many different interacting partners with a near-zero background8, 17
. However, the wavelength of light emitted during the enzymatic reaction of renilla luciferase with its substrate coelenterazine is in the range 480–510 nM, and this wavelength range sometimes incurs limitations of light absorption by different proteins in biological tissues (e.g., hemoglobin) when extending this system to imaging studies in small living animals.
To improve the absolute signal by increasing the protein-protein interaction assisted luciferase signal, we also reported a fusion protein strategy where a single vector encodes both interacting partners and the split reporters separated by linker sequences as a fusion protein18
. Whereas these published studies have already proven the utility of split bioluminescence reporters in studying protein-protein interactions, the focus of the current study was to use a combinatorial screening approach to identify new combinations of firefly luciferase fragments for studying protein-protein interactions with greater absolute signal and relatively low background that would be applicable to many different types of studies in cell culture and imaging small living animals.
We have collected over the last several years a library of N- and C- terminal fragments of firefly and renilla luciferase split reporter protein fragments in the course of developing and validating various split reporter strategies. In the current study, by applying a combinatorial screening approach to this library, we screened several combinations of N- and C- terminal firefly luciferase fragments with the interacting proteins FRB/FKBP12 and identified a new set of Nfluc and Cfluc fragments that have ideal properties of low background signal (low self-complementation) and a high signal after protein-protein interaction assisted complementation. In addition, this strategy also identified a number of combinations with and without overlapping regions and with and without self-complementing properties, in a single step. The selected combinations that produced the highest level of rapamycin induced luciferase signal were further evaluated with five other interacting protein combinations. An intramolecular folding strategy with the estrogen receptor and various ligands was also studied in cells and in mice with the identified optimal split reporters. The novel split reporters developed in this study will potentially increase the sensitivity and the generalizability of fragment-assisted complementation systems for studying protein-protein and other interactions in cells and small living animals.