BACKGROUND
it is estimated that there are about  <dig>  to  <dig>  protein-protein interactions  in human interactome  <cit> .
in order to discover and investigate ppi, exploration of ppi assays is considered highly important.
among various ppi assays, protein-fragment complementation assay  is a simple and user-friendly method  <cit> .
in pca, a reporter protein, such as a green fluorescent protein  variant  <cit>  and a luciferase  <cit>  is dissected to a split from, and each split reporter protein is fused to an interacting partner .
while the split reporter proteins have low reporter activity when they exist far apart , they reconstitute and recover the activity when the proteins come close due to the association of interacting partner .
in general, due to its low background activity, pca displays high signal to background ratio .
especially, bioluminescence-based pcas such as split-luciferase reporters shows reversible property  <cit> , in contrast to the irreversible nature of the reassembled gfp-like fluorescent proteins  <cit> .
in addition, since luciferases need no excitation light at shorter wavelength for their detection, the assay enables noninvasive and sensitive optical imaging in living cells and animals.
while ppi is conveniently assayed by pca in vivo and in cultured cells, it is often desirable to be performed also in vitro, to know whether the interaction is direct or not, since many ppi mediators, inhibitors and enhancers may exist in the complex cellular milieu.
to date, porter et al.
successfully utilized a cell-free transcription/translation system for performing firefly luciferase -based pca in vitro <cit> .
their probes can be prepared in shorter time period than the probes expressed in e. coli, yeast or mammalian cells.
however, the assay needs expensive cell-free lysate, and whose components always have a risk to affect ppi.
here we report a pca using purified interacting proteins fused with split fluc proteins, to detect ppi in a defined solution.
we also examined the stability of the probes, and the detectable distance between the interacting partners.
to the best of our knowledge, this will be the first investigation of pure in vitro ppi, based on fluc pca.
RESULTS
for the ppi to be investigated, we first selected a well-known interacting domain pair of fkbp <dig> and frb.
fkbp <dig> is a  <dig> kd domain of fk <dig> binding protein , which is able to associate with fkbp-rapamycin-associated protein  depending on an antibiotic, rapamycin   <cit> .
to this end, fkbp <dig> or frb gene was fused to the 5′ of fluc n-terminal  or c-terminal domain  gene, yielding four types of fusion protein genes .
the genes were inserted to pet32b vector, and the thioredoxin-fused proteins were expressed in the soluble fraction of e. coli bl <dig> and purified by an immobilized metal affinity chromatography .
the two interacting pairs  were mixed at  <dig> nm each, and equimolar rapamycin was added to the mixtures.
just after adding the two substrates atp and luciferin, the luminescent intensity was measured by a luminometer at  <dig>  s intervals for 4 s. as a result, the intensity of the interacting pairs added with rapamycin showed a marked increase.
in contrast, the pairs without rapamycin displayed very low luminescence .
in addition, non-interacting protein pairs , as well as each fusion protein alone exhibit very low luminescence even in the presence of rapamycin .
the results clearly showed that the ppi can be specifically detected with high s/b ratio using purified probes.
to assess the degree of spontaneous fluc reconstitution, the assay with one of the best interacting pairs frb/n and fkbp/c was performed at several probe concentrations .
at all the concentrations tested, the luminescent signals increased rapamycin-dependently .
the resulting maximum signal/background  ratio increased to as high as  <dig> when at the probe concentration increased up to  <dig> nm, while the ratio at  <dig> nm was slightly lower .
this was possibly because the high probe concentration rather promoted spontaneous fluc reconstitution without rapamycin.
when the rapamycin dose–response at  <dig> nm probe concentration was examined, the limit of detection was determined as  <dig> pm .
for fluc pca, several successful split sites are reported to date.
to investigate on this issue, another pair of n-terminal domain  together with the same c-terminal domain  was employed according to a previous attempt that gave good response in vivo   <cit> .
we could express and purify the new n-domain similarly , and obtained clear rapamycin-dependent signals at several concentrations of frb/n – fkbp/c pair .
however, compared with the frb/n – fkbp/c pair, the signal intensity was lower, and the resultant s/b ratio was rather unstable, albeit not low .
this was probably because of the instability of the n-domain , which is reflected to its degradation products observed in additional file 1: figure s1a.
since rapamycin-dependent fkbp-frb interaction is one of very strong protein-protein interactions  <cit> , another cellular interaction of mdm <dig> and p <dig> was investigated .
mdm <dig> oncoprotein is a cellular inhibitor of the p <dig> tumor suppressor in that it can bind the trans-activation domain of p <dig> and down-regulates its ability to activate transcription.
in certain cancers, mdm <dig> amplification is a common event and contributes to the inactivation of p <dig>  <cit> .
the probes fused to the trans-activation domain of p <dig> and mdm <dig>  were constructed in the same way as fkbp/c and frb/n  .
this time, both the signal intensity and s/b ratio elevated depending on the concentration of the probes .
then we investigated whether the in vitro fluc pca could detect the reversibility of interaction.
after we mixed the probes p53/c and mdm2/n at  <dig> nm each, nutlin- <dig>  a known inhibitor of p53-mdm <dig> interaction, was added and measured for the fluc activity .
the result clearly showed a nutlin- <dig> concentration dependent luminescence inhibition with calculated ic <dig> of 368 ±  <dig> nm.
although the value is higher than the reported one   <cit> , this might be due to the racemization of nutlin- <dig> used.
taken together, these results clearly showed the generality of the assay to detect ppis including transient interactions.
due to the nature of split enzyme, we reasoned that the instability of the purified probes could be a possible demerit of fluc-based pca.
to examine the stability of the probes, the mixtures of  <dig> nm fkbp/c and  <dig> nm frb/n with or without equimolar rapamycin were incubated at 37°c for 0–60 min, and applied to the luminescence assay .
as a result, the incubation resulted in a time-dependent decrease in luminescent intensity of the pair added with rapamycin .
as short as 15 min incubation resulted in the activity loss to less than one-third of that before incubation.
the longer incubation for 60 min resulted in virtually no signal difference between the pairs with and without rapamycin .
these results clearly indicate that the fluc pca probes have a limited stability in this purified milieu.
however, the results might also reflect their limited stability in vivo or in lysate, which is probably sequestered by the newly synthesized probes.
förster resonance energy transfer  is an excellent ppi detection method both in vitro and in vivo, and regarded as a standard tool in this area  <cit> .
however, fret has a fundamental limitation that it only occurs over rather short distance of several nm between the donor and the acceptor fluorophores, depending on förster distance r <dig>  which is determined a priori by the dye combination used  <cit> .
therefore, the interaction between proteins with large dimensions is a challenging task for fret, due to the possible long distance between the attached dyes including fluorescent proteins.
to compare fluc pca with fret, we examined the effect of distance between interacting partners.
previously, we introduced several helix-forming peptide linkers with various lengths between egfp and ebfp, and those linkers showed the ability to control the distance between interacting partners  <cit> .
according to this, a similar helix-forming linker, 4-repeats of asp-asp-ala-lys-lys , was inserted between fkbp <dig> and fluc c-terminal domain.
the sds polyacrylamide gel electrophoresis of the fusion proteins  suggests effective separation of fkbp <dig> and fluc c-terminal domain by the inserted linkers.
using  <dig> nm each mixture of fkbp/4 × ddakk/c, frb/n and rapamycin, ppi was detected with a high s/b ratio with a slight reduction compared with the pairs without helical linkers .
in addition, when a linker with  <dig> repeats of asp-asp-ala-lys-lys  was inserted between fkbp <dig> and fluc c-terminal domain, high s/b ratio was still maintained .
for a further examination, the 7th and 8th domains of fibronectin type iii  with a 7 nm n-c terminal distance was inserted between fkbp <dig> and fluc c-terminal domain, as a rigid linker.
from the sds-page of purified proteins, while the mobility of the reporter was lowered by the insertion of fn7- <dig>  no significant reduction in the yield and purity was observed .
ohashi et al.
reported that the insertion of fn7- <dig> domains between the fluorescent protein pair cypet and ypet results in barely detectable fret signal.
<cit> .
to confirm this, fn7- <dig> was inserted between fkbp <dig> and a brighter cfp variant cerulean  .
as expected, very weak fret signal derived of fkbp/fn7-8/cerulean and frb-fused ypet  was observed, although a distinct fret-derived peak was observed for the construct without fn7- <dig> insertion .
on the other hand, the pca pair of fkbp/fn7-8/c and frb/n at  <dig> nm displayed a moderate but significant s/b ratio, clearly showing its superiority over fret, although the observed rapamycin dependency was not so clear when reduced amounts  of the probes were used .
nevertheless, the fluc pca system was shown to work for detecting ppi over longer distance, which was not detectable by fret.
discussion
previously, fluc-based pca has been utilized as a sensitive tool to investigate ppi in vivo and in cultured cells.
however, considering possible disturbance by the co-existing components acting as ppi mediators, enhancers and inhibitors, ppi assay in vitro will also give us invaluable information.
furthermore, the in vitro assay has the possibility to study ppis between pathogen-derived cytotoxic proteins and host proteins, which are normally impossible to perform in vivo.
in this study, we showed that fluc pca also works with purified elements.
the system displayed high s/b ratio and unexpectedly rapid response .
in pca, the background signal due to nonspecific activation of the reporter can be a problem.
as in figure 2c, luminescent intensities of non-interacting pairs were slightly higher than those of single probe alone, probably reflecting the nonspecific reconstitution of split fluc.
however, when the specific ppi was induced by rapamycin, it resulted in markedly higher signal, giving high s/b ratio.
therefore, in practice the nonspecific signals hardly interfere with the ppi detection .
the only but not easily avoidable drawback of this system is the instability of probe as shown in additional file 2: figure s <dig>  apparently, the system is not very suitable for large-scale screening that needs longer sample preparation time.
however, proper cooling of the reagents and samples during the preparation period will surely reduce such inactivation.
therefore, we think that the system has a potential for low-cost and high-throughput drug screenings.
on the other hand, a distinct merit of this system is robustness to the interaction detection over longer distance.
in fact, insertion of either long helical linker or a rigid large protein domain did not deteriorate ppi detection, while fret signal was barely detectable when fn7- <dig> was inserted .
these results show a lesser limitation in distance between interacting partners in pca than in fret.
therefore, this system will be available for the interactions between large proteins that cannot be assayed by fret.
while the reason for observed nonspecific ppi signal remains unclear, it may be due to the interaction between the inserted sequence and the other probe.
in addition, we could observe the higher s/b ratio with the higher concentration of a probe set .
use of higher probe concentration is desirable to assay larger interacting partners.
the nearly straight shape of the kinetic curve observed might reflect the large dimension of fn7- <dig>  which would lower the reconstitution efficiency and subsequent luminescent reaction rate.
collectively, considering the demerits and the merits, fluc pca using purified elements enables us a rapid and convenient detection of direct ppi with high confidence.
CONCLUSIONS
the feasibility of fluc pca in vitro using purified elements was demonstrated for the first time.
the assay was found to have high s/b ratio, and allow the detection of larger size of interacting partner than detectable by fret.
in spite of relatively weak thermostability, the assay will be applicable to handy detection of various ppis in vitro including in vitro diagnostics and drug screening.

 it is estimated that there are about to protein-protein interactions in human interactome. in order to discover and investigate ppi, exploration of ppi assays is considered highly important. among various ppi assays, protein-fragment complementation assay is a simple and user-friendly method. in pca, a reporter protein, such as a green fluorescent protein variant and a luciferase is dissected to a split from, and each split reporter protein is fused to an interacting partner. while the split reporter proteins have low reporter activity when they exist far apart, they reconstitute and recover the activity when the proteins come close due to the association of interacting partner. in general, due to its low background activity, pca displays high signal to background ratio. especially, bioluminescence-based pcas such as split-luciferase reporters shows reversible property, in contrast to the irreversible nature of the reassembled gfp-like fluorescent proteins. in addition, since luciferases need no excitation light at shorter wavelength for their detection, the assay enables noninvasive and sensitive optical imaging in living cells and animals. while ppi is conveniently assayed by pca in vivo and in cultured cells, it is often desirable to be performed also in vitro, to know whether the interaction is direct or not, since many ppi mediators, inhibitors and enhancers may exist in the complex cellular milieu. to date, porter et al. successfully utilized a cell-free transcription/translation system for performing firefly luciferase -based pca in vitro. their probes can be prepared in shorter time period than the probes expressed in e. coli, yeast or mammalian cells. however, the assay needs expensive cell-free lysate, and whose components always have a risk to affect ppi. here we report a pca using purified interacting proteins fused with split fluc proteins, to detect ppi in a defined solution. we also examined the stability of the probes, and the detectable distance between the interacting partners. to the best of our knowledge, this will be the first investigation of pure in vitro ppi, based on fluc pca.  for the ppi to be investigated, we first selected a well-known interacting domain pair of fkbp and frb. fkbp is a kd domain of fk binding protein, which is able to associate with fkbp-rapamycin-associated protein depending on an antibiotic, rapamycin. to this end, fkbp or frb gene was fused to the 5′ of fluc n-terminal or c-terminal domain gene, yielding four types of fusion protein genes. the genes were inserted to pet32b vector, and the thioredoxin-fused proteins were expressed in the soluble fraction of e. coli bl and purified by an immobilized metal affinity chromatography. the two interacting pairs were mixed at nm each, and equimolar rapamycin was added to the mixtures. just after adding the two substrates atp and luciferin, the luminescent intensity was measured by a luminometer at  s intervals for 4 s. as a result, the intensity of the interacting pairs added with rapamycin showed a marked increase. in contrast, the pairs without rapamycin displayed very low luminescence. in addition, non-interacting protein pairs, as well as each fusion protein alone exhibit very low luminescence even in the presence of rapamycin. the results clearly showed that the ppi can be specifically detected with high s/b ratio using purified probes. to assess the degree of spontaneous fluc reconstitution, the assay with one of the best interacting pairs frb/n and fkbp/c was performed at several probe concentrations. at all the concentrations tested, the luminescent signals increased rapamycin-dependently. the resulting maximum signal/background ratio increased to as high as when at the probe concentration increased up to nm, while the ratio at nm was slightly lower. this was possibly because the high probe concentration rather promoted spontaneous fluc reconstitution without rapamycin. when the rapamycin dose–response at nm probe concentration was examined, the limit of detection was determined as pm. for fluc pca, several successful split sites are reported to date. to investigate on this issue, another pair of n-terminal domain together with the same c-terminal domain was employed according to a previous attempt that gave good response in vivo. we could express and purify the new n-domain similarly, and obtained clear rapamycin-dependent signals at several concentrations of frb/n – fkbp/c pair. however, compared with the frb/n – fkbp/c pair, the signal intensity was lower, and the resultant s/b ratio was rather unstable, albeit not low. this was probably because of the instability of the n-domain, which is reflected to its degradation products observed in additional file 1: figure s1a. since rapamycin-dependent fkbp-frb interaction is one of very strong protein-protein interactions, another cellular interaction of mdm and p was investigated. mdm oncoprotein is a cellular inhibitor of the p tumor suppressor in that it can bind the trans-activation domain of p and down-regulates its ability to activate transcription. in certain cancers, mdm amplification is a common event and contributes to the inactivation of p. the probes fused to the trans-activation domain of p and mdm were constructed in the same way as fkbp/c and frb/n. this time, both the signal intensity and s/b ratio elevated depending on the concentration of the probes. then we investigated whether the in vitro fluc pca could detect the reversibility of interaction. after we mixed the probes p53/c and mdm2/n at nm each, nutlin- a known inhibitor of p53-mdm interaction, was added and measured for the fluc activity. the result clearly showed a nutlin- concentration dependent luminescence inhibition with calculated ic of 368 ±  nm. although the value is higher than the reported one, this might be due to the racemization of nutlin- used. taken together, these results clearly showed the generality of the assay to detect ppis including transient interactions. due to the nature of split enzyme, we reasoned that the instability of the purified probes could be a possible demerit of fluc-based pca. to examine the stability of the probes, the mixtures of nm fkbp/c and nm frb/n with or without equimolar rapamycin were incubated at 37°c for 0–60 min, and applied to the luminescence assay. as a result, the incubation resulted in a time-dependent decrease in luminescent intensity of the pair added with rapamycin. as short as 15 min incubation resulted in the activity loss to less than one-third of that before incubation. the longer incubation for 60 min resulted in virtually no signal difference between the pairs with and without rapamycin. these results clearly indicate that the fluc pca probes have a limited stability in this purified milieu. however, the results might also reflect their limited stability in vivo or in lysate, which is probably sequestered by the newly synthesized probes. förster resonance energy transfer is an excellent ppi detection method both in vitro and in vivo, and regarded as a standard tool in this area. however, fret has a fundamental limitation that it only occurs over rather short distance of several nm between the donor and the acceptor fluorophores, depending on förster distance r which is determined a priori by the dye combination used. therefore, the interaction between proteins with large dimensions is a challenging task for fret, due to the possible long distance between the attached dyes including fluorescent proteins. to compare fluc pca with fret, we examined the effect of distance between interacting partners. previously, we introduced several helix-forming peptide linkers with various lengths between egfp and ebfp, and those linkers showed the ability to control the distance between interacting partners. according to this, a similar helix-forming linker, 4-repeats of asp-asp-ala-lys-lys, was inserted between fkbp and fluc c-terminal domain. the sds polyacrylamide gel electrophoresis of the fusion proteins suggests effective separation of fkbp and fluc c-terminal domain by the inserted linkers. using nm each mixture of fkbp/4 × ddakk/c, frb/n and rapamycin, ppi was detected with a high s/b ratio with a slight reduction compared with the pairs without helical linkers. in addition, when a linker with repeats of asp-asp-ala-lys-lys was inserted between fkbp and fluc c-terminal domain, high s/b ratio was still maintained. for a further examination, the 7th and 8th domains of fibronectin type iii with a 7 nm n-c terminal distance was inserted between fkbp and fluc c-terminal domain, as a rigid linker. from the sds-page of purified proteins, while the mobility of the reporter was lowered by the insertion of fn7- no significant reduction in the yield and purity was observed. ohashi et al. reported that the insertion of fn7- domains between the fluorescent protein pair cypet and ypet results in barely detectable fret signal.. to confirm this, fn7- was inserted between fkbp and a brighter cfp variant cerulean. as expected, very weak fret signal derived of fkbp/fn7-8/cerulean and frb-fused ypet was observed, although a distinct fret-derived peak was observed for the construct without fn7- insertion. on the other hand, the pca pair of fkbp/fn7-8/c and frb/n at nm displayed a moderate but significant s/b ratio, clearly showing its superiority over fret, although the observed rapamycin dependency was not so clear when reduced amounts of the probes were used. nevertheless, the fluc pca system was shown to work for detecting ppi over longer distance, which was not detectable by fret. discussion previously, fluc-based pca has been utilized as a sensitive tool to investigate ppi in vivo and in cultured cells. however, considering possible disturbance by the co-existing components acting as ppi mediators, enhancers and inhibitors, ppi assay in vitro will also give us invaluable information. furthermore, the in vitro assay has the possibility to study ppis between pathogen-derived cytotoxic proteins and host proteins, which are normally impossible to perform in vivo. in this study, we showed that fluc pca also works with purified elements. the system displayed high s/b ratio and unexpectedly rapid response. in pca, the background signal due to nonspecific activation of the reporter can be a problem. as in figure 2c, luminescent intensities of non-interacting pairs were slightly higher than those of single probe alone, probably reflecting the nonspecific reconstitution of split fluc. however, when the specific ppi was induced by rapamycin, it resulted in markedly higher signal, giving high s/b ratio. therefore, in practice the nonspecific signals hardly interfere with the ppi detection. the only but not easily avoidable drawback of this system is the instability of probe as shown in additional file 2: figure s apparently, the system is not very suitable for large-scale screening that needs longer sample preparation time. however, proper cooling of the reagents and samples during the preparation period will surely reduce such inactivation. therefore, we think that the system has a potential for low-cost and high-throughput drug screenings. on the other hand, a distinct merit of this system is robustness to the interaction detection over longer distance. in fact, insertion of either long helical linker or a rigid large protein domain did not deteriorate ppi detection, while fret signal was barely detectable when fn7- was inserted. these results show a lesser limitation in distance between interacting partners in pca than in fret. therefore, this system will be available for the interactions between large proteins that cannot be assayed by fret. while the reason for observed nonspecific ppi signal remains unclear, it may be due to the interaction between the inserted sequence and the other probe. in addition, we could observe the higher s/b ratio with the higher concentration of a probe set. use of higher probe concentration is desirable to assay larger interacting partners. the nearly straight shape of the kinetic curve observed might reflect the large dimension of fn7- which would lower the reconstitution efficiency and subsequent luminescent reaction rate. collectively, considering the demerits and the merits, fluc pca using purified elements enables us a rapid and convenient detection of direct ppi with high confidence.  the feasibility of fluc pca in vitro using purified elements was demonstrated for the first time. the assay was found to have high s/b ratio, and allow the detection of larger size of interacting partner than detectable by fret. in spite of relatively weak thermostability, the assay will be applicable to handy detection of various ppis in vitro including in vitro diagnostics and drug screening. 
this would be the first demonstration of fluc pca in vitro, which has a distinct advantage over other ppi assays. when fn7- domains that was shown to abrogate gfp mutant-based fret was inserted between split fluc and fkbp as a rigid linker, it still showed some response, suggesting less limitation in interacting partner’s size. to date, successful applications of firefly luciferase -based pca have been reported in vivo, in cultured cells and in cell-free lysate, owing to its high sensitivity, high signal-to-background ratio, and reversible response. protein-fragment complementation assay is one of the most widely used methods to detect ppi, as well as förster resonance energy transfer. our system enables detection of direct ppis without risk of perturbation by ppi mediators in the complex cellular milieu. fluc pca using purified components will enable a rapid and handy detection of ppis with high s/b ratio, avoiding the effects of concomitant components. when the proteins were used for pca to detect rapamycin-dependent ppi, they enabled a rapid detection of ppi with high s/b ratio.
 human interactome is predicted to contain to protein-protein interactions,. protein-fragment complementation assay is one of the most widely used methods to detect ppi, as well as förster resonance energy transfer. to date, successful applications of firefly luciferase -based pca have been reported in vivo, in cultured cells and in cell-free lysate, owing to its high sensitivity, high signal-to-background ratio, and reversible response. here we show the assay also works with purified proteins with unexpectedly rapid kinetics.  split fluc fragments both fused with a rapamycin-dependently interacting protein pair were made and expressed in e. coli system, and purified to homogeneity. when the proteins were used for pca to detect rapamycin-dependent ppi, they enabled a rapid detection of ppi with high s/b ratio. when fn7- domains that was shown to abrogate gfp mutant-based fret was inserted between split fluc and fkbp as a rigid linker, it still showed some response, suggesting less limitation in interacting partner’s size. finally, the stability of the probe was investigated. preincubation of the probes at degreec up to 1 h showed marked decrease of the luminescent signal to %, showing the limited stability of this system.  fluc pca using purified components will enable a rapid and handy detection of ppis with high s/b ratio, avoiding the effects of concomitant components. although the system might not be suitable for large-scale screening due to its limited stability, it can detect an interaction over larger distance than by fret. this would be the first demonstration of fluc pca in vitro, which has a distinct advantage over other ppi assays. our system enables detection of direct ppis without risk of perturbation by ppi mediators in the complex cellular milieu. protein-protein interactionfirefly luciferasebioluminescenceprotein fragment complementation assaythermostabilityin vitro diagnostics 