BACKGROUND
clostridium difficile is a gram-positive, spore-forming anaerobic bacillus.
it is the most common cause of nosocomial antibiotic-associated diarrhea and the etiologic agent of pseudomembranous colitis  <cit> .
antibiotic usage results in a reduction of commensal microflora in the gut, which permits c. difficile to proliferate more extensively, leading to the production of toxins  <cit> .
c. difficile associated diarrhea  include a range of symptoms varying from mild diarrhea to severe fulminate lethal disease  <cit> .
recent outbreaks of highly virulent c. difficile strains  <cit>  have increased the urgency to devote greater resources towards the understanding of the molecular, genetic, and biochemical basis for the pathogenesis, with a view to use such information to develop novel preventative and treatment modalities.
two exotoxins produced by toxigenic c. difficile, toxin a  and toxin b , are most extensively studied and thought to be major virulent factors of cdad  <cit> .
tcda  and tcdb  belong to the large clostridial cytotoxin  family and share 49% amino acid identity  <cit> .
the two toxins have a similar structure containing a putative receptor binding domain , a transmembrane domain , and a glucosyltransferase domain  <cit> .
after receptor-mediated internalization and intracellular cleavage, the toxins glucosylate members of the rho-rac family of small gtpases at a specific threonine residue in host intestinal epithelial cells, leading to alterations in the actin cytoskeleton, massive fluid secretion, acute inflammation, and necrosis of the colonic mucosa  <cit> .
purified tcda possesses potent enterotoxic and pro-inflammatory activity, as determined in ligated intestinal loop studies in animals  <cit> .
tcda is also cytotoxic to cultured cells in a picomolar to nanomolar range.
tcdb, more cytotoxic to cultured cells than tcda, was previously reported to exhibit no enterotoxic activity in animals  <cit> , but recent studies have found enterotoxic and proinflammatory activities in human intestinal xenografts in severe combined immunodeficient  mice  <cit> .
furthermore, the tcda-b+ c. difficile strains are responsible for pseudomembranous colitis in some patients  <cit> .
it is desirable to obtain relatively pure and biologically active tcda and tcdb for studying the pathogenesis of cdad and host immune response to the infection and for generating immunological tools for research and clinical diagnosis.
the native toxins are usually purified from toxigenic c. difficile culture supernatant, which involves multiple steps and the purity is often unsatisfactory  <cit> .
attempts have been made to clone and express c. difficile toxins in escherichia coli  <cit> , but it is unclear whether or not purified toxins were obtained from the bacterial lysate in these studies.
the gram-positive bacillus megaterium expression system may offer an alternative for the expression of c. difficile toxins due to several advantages over the e. coli system, including the lack of alkaline proteases activity and endotoxin lps, capable of secreting expressed heterologous protein into the medium  <cit> .
although the expression level was low, burger et al  <cit>  were the first to successfully express and obtain the purified recombinant tcda in b. megaterium.
in this study, we have expressed the full-length of both tcda and tcdb in b. megaterium.
we were able to obtain an average of  <dig> –  <dig> mg of highly purified recombinant proteins from one liter of total bacterial culture.
both recombinant tcda and tcdb were biologically active similar to their native purified toxins.
RESULTS
cloning and expression of the recombinant toxins
after enzyme  digestion of phis <dig> vector and pcr products, the ligation was set up and the mixture was incubated at 4°c for  <dig> days.
the transformation was carried out following the standard protocol and more than  <dig> bacterial colonies were picked up.
the plasmids  from these colonies were subjected to screening by digestion with a variety of enzymes.
phis-tcdb from multiple clones was used to transform b. megaterium protoplasts, and tetracycline-resistant clones were picked up.
after induction with xylose, the bacterial crude extracts from approximately  <dig> different transformed clones were subjected to screening for activity causing rounding of cultured cells.
the clones with high activities were selected and a coomassie-staining gel showed expression of a protein of about  <dig> kda when xylose was added for induction .
this protein was purified by ni-affinity column from a total crude extract, resulting in a major band with molecular weight about  <dig> kda and some weak contaminant bands of lower molecular weights .
a further step of ion-exchange purification resulted in a highly pure  <dig> kda-protein without any visible contaminant bands on coomassie-stained gels .
western blot analysis using specific antibodies against his-tags  and toxin b  identified the  <dig> kda protein from b. megaterium lysate as the recombinant his-tagged toxin b .
from one liter of bacterial culture, approximately  <dig> mg of rtcdb was obtained.
the phis-tcdb used to transform the b. megaterium was subjected to dna sequencing for the verification of tcdb gene, and no mutation was found .
b. megaterium has a secretory pathway  <cit> , which transports the expressed heterologous proteins into the medium.
because the native toxins are secreted into medium by toxigenic c. difficile, we attempted the expression of rtcdb in a secretory form.
the dna sequence encoding a 28-amino-acid signal peptide derived from b. megaterium extracellular esterase lipa  <cit>  was synthesized and inserted at the 5' of tcdb.
the resulting phis-sp-tcdb was transformed into b. megaterium.
figure 1d shows the presence of a  <dig> kda band from the concentrated bacterial culture supernatant on a coomassie stained sds-page gel .
western blot and cytotoxicity assays confirmed the expression of the secretory form of rtcdb.
the expression level of this secretory form was, however, very low and we were unable to obtain a sufficient amount of purified rtcdb from the supernatant.
a similar strategy was utilized to clone and express recombinant tcda.
on a coomassie stained sds-page gel of total bacterial lysate, a clear protein band of  <dig> kda appeared only upon induction with xylose in phis-tcda-transformed b. megaterium .
his-tag combined with thyroglobulin affinity purifications resulted in a single strong band , which was confirmed to be tcda by western blot analysis .
the phis-tcda used to transform the b. megaterium was subjected to dna sequencing for the verification of tcda gene, and no mutation was found .
cytopathic effect of recombinant toxins
we compared the cytopathic effects of recombinant and native toxins on cultured human epithelial ht- <dig>  and mouse intestinal epithelial ct <dig> cells .
rtcda  at  <dig> or  <dig> ng/ml caused ct <dig> cell rounding similarly to native tcda .
both rtcdb and native tcdb were more potent, capable of causing cell rounding at a much lower concentration than those of rtcda or native tcda .
after xylose induction, both supernatant  and total cell lysate  from phis-sp-tcdb transformed b. megaterium caused cell rounding, whereas the bacterial culture supernatants from phis-tcdb transformed b. megaterium with  or without  xylose induction did not cause cell rounding.
the same concentration of xylose in bhi medium did not cause any cell rounding , suggesting the effects were specifically caused by the secreted toxin.
furthermore, the cytopathic effects of both rtcda and native tcda were blocked completely by a rabbit polyclonal antibody against tcda .
cytotoxic effect of recombinant toxins
it has been demonstrated that both tcda and tcdb induce intestinal epithelial cells to undergo apoptosis  <cit> .
we next compared the cytotoxicity of recombinant toxins to their native counterparts on cultured epithelial cells.
as shown in figure 4a, both native and recombinant tcda induced a comparable and dose-dependent cell death of ct <dig> cells.
similarly, the exposure of ct <dig> cells to rtcdb induced a comparable cell death with ntcdb as determined by mtt assay .
these data indicate that the recombinant tcda and tcdb have similar cytotoxic activity to their native counterparts in ct <dig> cells.
glucosylation of rac <dig> by recombinant toxins
c. difficile tcda and tcdb target host cell rho gtpase by glucosylating these proteins at the specific thr <dig>  or thr <dig>  residues  <cit> .
to determine whether recombinant toxins can glucosylate rac <dig> protein of host cells, mouse intestinal epithelial ct <dig> cells were treated with recombinant or native tcda and tcdb for  <dig> hr.
western blot showed that both rtcda and ntcda treatment induced a dose-dependent, reduced recognition of rac <dig> by monoclonal antibody  that recognizes non-glucosylated rac <dig>  <cit>  .
the reduced recognition of rac <dig> was not the results of protein degradation, because total rac <dig> protein remained unchanged as determined by western blot with an antibody  recognizing both glucosylated and unmodified rac <dig> .
both rtcda and ntcda at  <dig> ng/ml resulted in a complete glucosylation of rac <dig> whereas  <dig> or  <dig>  ng/ml of tcda led to a partial glucosylation.
rtcdb or ntcdb exposure resulted in a completely glucosylation of rac <dig> at a dose range from  <dig>  –  <dig> ng/ml , indicating that the glucosyltransferase activity of tcdb was more potent than tcda.
these results were consistent with the activity of native c. difficile toxins in which tcdb is more toxic to cultured cells than tcda.
the treatment of rtcda and rtcdb also led to the glucosylation of rac <dig> in raw  <dig>  and cho cells .
both tcda and tcdb are thought to bind to specific cellular receptor, which mediate their cellular uptake through endocytosis  <cit> .
lysosomotropic agents such as chloroquine and nh4cl can inhibit toxin-mediated cytotoxicity, suggesting that the endosomal acidification is involved in toxin intracellular trafficking  <cit> .
to determine whether or not the cellular activity of the recombinant toxins also require endosomal acidification, ct <dig> cells were pre-incubated with nh4cl for  <dig> min before the toxin exposure.
while the exposure of ct <dig> cells to rtcda or rtcdb induced the glucosylation of rac <dig>  pretreatment of cells with ammonium chloride completely blocked such an activity .
disruption of tight junctions of intestinal epithelial cells by recombinant toxins
it is known that tcda and tcdb alter the structure of intestinal epithelia by disrupting tight junctions  <cit> .
we investigated whether or not the recombinant toxins we generated have similar activities.
human intestinal epithelial hct- <dig> cells were cultured in a transwell for  <dig> to  <dig> days until transepithelial resistance  reached  <dig> Ω*cm <dig> and tight junctions were formed .
rtcda did not alter cell-cell connections of hct- <dig> cells within  <dig> hr of treatment  but disrupted tight junctions after  <dig> hr of treatment .
rtcdb, at the same concentration with rtcda, completely disrupted the tight junctions of hct- <dig> within  <dig> hr of treatment .
discussion
cdad is largely attributed to the bacterial exotoxins tcda and tcdb, although other virulence factors probably also play some roles  <cit> .
both toxins glucosylate rho gtpase family proteins, leading to their inactivation  <cit> .
to study the pathogenesis of cdad and host immune response to the toxins, and to use the toxins as a research tool for signal transduction involving rho gtpases, it is necessary to obtain large quantities of highly purified active tcda and tcdb.
native toxins can be purified from toxigenic c. difficile culture supernatant.
the purification process however usually involves multiple steps such as ultrafiltration, ammonium sulfate precipitation, and ion-exchange chromatography  <cit> .
in the case of tcda, affinity purification through thyroglobulin is available and relatively pure tcda can be obtained .
tcdb however is often contaminated with some unknown substance even after extensive purification steps .
gene engineering to generate recombinant toxins seems to be a logical approach for obtaining large quantities of pure toxin proteins.
the e. coli system is popular and is widely used for the expression of recombinant proteins.
several studies have reported to have cloned and expressed active c. difficile toxins in e. coli, but failed to demonstrate that the purified toxins were obtained from bacterial extracts  <cit> .
burger et al was the first to report a successful expression of recombinant tcda using bacillus megaterium system  <cit> .
recombinant tcdb, however, has not yet been stably expressed.
in this study, we were successfully expressing a full-length recombinant tcdb in b. megaterium and greatly enhanced the expression level of recombinant tcda.
because e. coli expression system is widely used and easy-to-handle, we have utilized various e. coli expression vector systems to clone and express both toxins and found that both recombinant tcda and tcdb were expressed in e. coli but we were unable to obtain the purified toxins from the bacterial lysate .
the large size of the heterologous proteins may possibly be prone to protease digestion, and intrinsic protease activities of these toxins  <cit>  may also contribute to the instability.
another possible reason for the inability to obtain purified recombinant toxins from e. coli is that the expression level was too low.
in fact, large proteins are difficult to express at high levels in e. coli.
furthermore, an unusually high content of at  in these clostridial gene sequences and a different codon -usage make them particularly difficult to express at a high level in e. coli  <cit> .
there are several advantages associated with b. megaterium expression system including lack of alkaline phosphatase and endotoxin lps.
more importantly, b. megaterium has been successfully used to express rtcda  <cit> , we therefore decided to use this system.
initially, we followed the reported cloning strategy but we were unsuccessful.
this might be due to the extremely low ligation efficiency  <cit>  when putting several fragments together during the cloning.
we then changed our strategy to a direct pcr amplification of whole toxin genes.
however, this strategy was associated with a potential risk that pcr might introduce mutations due to the large size of the genes .
because of the large size of both genes, it was impossible to sequence all constructs in order to identify the correct clones.
we therefore took a different approach, screening for recombinant protein expression and cytotoxicity.
we screened as many colonies as possible  after ligation  and then transformed these constructed shuttle vectors into b. megaterium protoplasts.
the resulting colonies were subjected to screening again by examining the cytotoxic activity in bacterial crude extracts.
the specific expression of recombinant toxins from the positive colonies  was then determined by western blot.
the plasmids encoding tcda or tcdb respectively were finally subjected to dna sequencing and no single mutation was found in either of the toxin genes.
the average yields of recombinant tcda and tcdb were about  <dig> –  <dig> mg/liter of bacterial culture, which were much higher than that of native toxins from c. difficile culture, and up to 20-fold higher than the amount of rtcda reported by burger et al  <cit> .
the fact that we used a different shuttle vector from that of burger et al  <cit>  might explain our higher yields of recombinant proteins.
in addition, we have found out a twelve- to sixteen-hour period of induction with xylose yielded more recombinant proteins than five hours as suggested by the manufacturer.
the other possible explanation was that the tcda gene was fused into orf <dig> of the vector in the construct reported by burger et al  <cit> , resulting a modified n-terminus of recombinant tcda.
this modification might affect the efficiency of protein expression and/or its stability.
we inserted the tcda or tcdb gene into the bsrgi site and the protein translation directly started from toxin gene, resulting in recombinant toxins without any n-terminal modification.
to facilitate purification, a his <dig> tag was fused to the c-terminus of both toxins.
one-step of ni-affinity chromatography resulted in approximately 70–80% purity of recombinant toxins as determined by coomassie staining of sds-page .
to achieve a greater purity, an additional step was introduced after ni-affinity chromatography.
for rtcda, thyroglobulin affinity purification was used.
rtcdb was further purified by an anion-exchange fractionation .
these additional purification steps for rtcda and rtcdb, respectively, led to highly pure recombinant toxins.
no additional band was seen on a coomassie-stained gel .
in addition, there were no detectable tlr <dig> or tlr <dig> stimulants within the purified recombinant toxins as determined by sensitive bioassays .
this is important because any use of the impure proteins to study host immune response or signaling transduction would possibly result in unintended consequences.
unlike e. coli, a protein secretory pathway is present in b. megaterium  <cit> .
a 28-amino-acid signal peptide of b. megaterium extracellular esterase lipa can direct the transport of expressed heterologous proteins into medium and the sequence is removed by a protease after secretion  <cit> .
because of the nature of exotoxins of native toxins and relatively easy purification from supernatant without the need for sonication or other means of cell disruptions, we sought to express rtcdb extracellularly.
after inserting the gene of the leader signaling peptide at the 5' of tcdb, we were able to generate active secretory rtcdb  and coomassie staining of concentrated supernatant on a sds-page gel and western blot showed rtcdb band .
however, for some unknown reasons, the expression level of rtcdb was significantly decreased when the leader sequence was introduced to the construct.
in addition, we found the secretion was very inefficient and the majority of toxins were still trapped inside of the bacteria, since the total bacterial lysate contained the majority of toxin activity .
this secretory pathway may not be efficient for such a large size of protein.
the low amount of rtcdb in the medium might also be due to the inefficient refolding after their extracellular transport.
CONCLUSIONS
in this study, we have successfully expressed the full-length of recombinant c. difficile toxin a and b proteins in b. megaterium and greatly improved the yield of the recombinant toxins.
we were able to obtain  <dig> –  <dig> mg of highly purified rtcda or rtcdb from one liter of bacterial culture.
these recombinant toxins had similar biological activities with their native counterparts.
we believe the recombinant toxin proteins will provide an invaluable tool for studying the pathogenesis of c. difficile associated disease and host immune response to toxins, as well as tools for studying the signaling transduction involving rho gtpase family proteins.

 clostridium difficile is a gram-positive, spore-forming anaerobic bacillus. it is the most common cause of nosocomial antibiotic-associated diarrhea and the etiologic agent of pseudomembranous colitis. antibiotic usage results in a reduction of commensal microflora in the gut, which permits c. difficile to proliferate more extensively, leading to the production of toxins. c. difficile associated diarrhea include a range of symptoms varying from mild diarrhea to severe fulminate lethal disease. recent outbreaks of highly virulent c. difficile strains have increased the urgency to devote greater resources towards the understanding of the molecular, genetic, and biochemical basis for the pathogenesis, with a view to use such information to develop novel preventative and treatment modalities. two exotoxins produced by toxigenic c. difficile, toxin a and toxin b, are most extensively studied and thought to be major virulent factors of cdad. tcda and tcdb belong to the large clostridial cytotoxin family and share 49% amino acid identity. the two toxins have a similar structure containing a putative receptor binding domain, a transmembrane domain, and a glucosyltransferase domain. after receptor-mediated internalization and intracellular cleavage, the toxins glucosylate members of the rho-rac family of small gtpases at a specific threonine residue in host intestinal epithelial cells, leading to alterations in the actin cytoskeleton, massive fluid secretion, acute inflammation, and necrosis of the colonic mucosa. purified tcda possesses potent enterotoxic and pro-inflammatory activity, as determined in ligated intestinal loop studies in animals. tcda is also cytotoxic to cultured cells in a picomolar to nanomolar range. tcdb, more cytotoxic to cultured cells than tcda, was previously reported to exhibit no enterotoxic activity in animals, but recent studies have found enterotoxic and proinflammatory activities in human intestinal xenografts in severe combined immunodeficient mice. furthermore, the tcda-b+ c. difficile strains are responsible for pseudomembranous colitis in some patients. it is desirable to obtain relatively pure and biologically active tcda and tcdb for studying the pathogenesis of cdad and host immune response to the infection and for generating immunological tools for research and clinical diagnosis. the native toxins are usually purified from toxigenic c. difficile culture supernatant, which involves multiple steps and the purity is often unsatisfactory. attempts have been made to clone and express c. difficile toxins in escherichia coli, but it is unclear whether or not purified toxins were obtained from the bacterial lysate in these studies. the gram-positive bacillus megaterium expression system may offer an alternative for the expression of c. difficile toxins due to several advantages over the e. coli system, including the lack of alkaline proteases activity and endotoxin lps, capable of secreting expressed heterologous protein into the medium. although the expression level was low, burger et al were the first to successfully express and obtain the purified recombinant tcda in b. megaterium. in this study, we have expressed the full-length of both tcda and tcdb in b. megaterium. we were able to obtain an average of – mg of highly purified recombinant proteins from one liter of total bacterial culture. both recombinant tcda and tcdb were biologically active similar to their native purified toxins.  cloning and expression of the recombinant toxins after enzyme digestion of phis vector and pcr products, the ligation was set up and the mixture was incubated at 4°c for days. the transformation was carried out following the standard protocol and more than bacterial colonies were picked up. the plasmids from these colonies were subjected to screening by digestion with a variety of enzymes. phis-tcdb from multiple clones was used to transform b. megaterium protoplasts, and tetracycline-resistant clones were picked up. after induction with xylose, the bacterial crude extracts from approximately different transformed clones were subjected to screening for activity causing rounding of cultured cells. the clones with high activities were selected and a coomassie-staining gel showed expression of a protein of about kda when xylose was added for induction. this protein was purified by ni-affinity column from a total crude extract, resulting in a major band with molecular weight about kda and some weak contaminant bands of lower molecular weights. a further step of ion-exchange purification resulted in a highly pure kda-protein without any visible contaminant bands on coomassie-stained gels. western blot analysis using specific antibodies against his-tags and toxin b identified the kda protein from b. megaterium lysate as the recombinant his-tagged toxin b. from one liter of bacterial culture, approximately mg of rtcdb was obtained. the phis-tcdb used to transform the b. megaterium was subjected to dna sequencing for the verification of tcdb gene, and no mutation was found. b. megaterium has a secretory pathway, which transports the expressed heterologous proteins into the medium. because the native toxins are secreted into medium by toxigenic c. difficile, we attempted the expression of rtcdb in a secretory form. the dna sequence encoding a 28-amino-acid signal peptide derived from b. megaterium extracellular esterase lipa was synthesized and inserted at the 5' of tcdb. the resulting phis-sp-tcdb was transformed into b. megaterium. figure 1d shows the presence of a kda band from the concentrated bacterial culture supernatant on a coomassie stained sds-page gel. western blot and cytotoxicity assays confirmed the expression of the secretory form of rtcdb. the expression level of this secretory form was, however, very low and we were unable to obtain a sufficient amount of purified rtcdb from the supernatant. a similar strategy was utilized to clone and express recombinant tcda. on a coomassie stained sds-page gel of total bacterial lysate, a clear protein band of kda appeared only upon induction with xylose in phis-tcda-transformed b. megaterium. his-tag combined with thyroglobulin affinity purifications resulted in a single strong band, which was confirmed to be tcda by western blot analysis. the phis-tcda used to transform the b. megaterium was subjected to dna sequencing for the verification of tcda gene, and no mutation was found. cytopathic effect of recombinant toxins we compared the cytopathic effects of recombinant and native toxins on cultured human epithelial ht- and mouse intestinal epithelial ct cells. rtcda at or ng/ml caused ct cell rounding similarly to native tcda. both rtcdb and native tcdb were more potent, capable of causing cell rounding at a much lower concentration than those of rtcda or native tcda. after xylose induction, both supernatant and total cell lysate from phis-sp-tcdb transformed b. megaterium caused cell rounding, whereas the bacterial culture supernatants from phis-tcdb transformed b. megaterium with or without xylose induction did not cause cell rounding. the same concentration of xylose in bhi medium did not cause any cell rounding, suggesting the effects were specifically caused by the secreted toxin. furthermore, the cytopathic effects of both rtcda and native tcda were blocked completely by a rabbit polyclonal antibody against tcda. cytotoxic effect of recombinant toxins it has been demonstrated that both tcda and tcdb induce intestinal epithelial cells to undergo apoptosis. we next compared the cytotoxicity of recombinant toxins to their native counterparts on cultured epithelial cells. as shown in figure 4a, both native and recombinant tcda induced a comparable and dose-dependent cell death of ct cells. similarly, the exposure of ct cells to rtcdb induced a comparable cell death with ntcdb as determined by mtt assay. these data indicate that the recombinant tcda and tcdb have similar cytotoxic activity to their native counterparts in ct cells. glucosylation of rac by recombinant toxins c. difficile tcda and tcdb target host cell rho gtpase by glucosylating these proteins at the specific thr or thr residues. to determine whether recombinant toxins can glucosylate rac protein of host cells, mouse intestinal epithelial ct cells were treated with recombinant or native tcda and tcdb for hr. western blot showed that both rtcda and ntcda treatment induced a dose-dependent, reduced recognition of rac by monoclonal antibody that recognizes non-glucosylated rac. the reduced recognition of rac was not the results of protein degradation, because total rac protein remained unchanged as determined by western blot with an antibody recognizing both glucosylated and unmodified rac. both rtcda and ntcda at ng/ml resulted in a complete glucosylation of rac whereas or ng/ml of tcda led to a partial glucosylation. rtcdb or ntcdb exposure resulted in a completely glucosylation of rac at a dose range from – ng/ml, indicating that the glucosyltransferase activity of tcdb was more potent than tcda. these results were consistent with the activity of native c. difficile toxins in which tcdb is more toxic to cultured cells than tcda. the treatment of rtcda and rtcdb also led to the glucosylation of rac in raw and cho cells. both tcda and tcdb are thought to bind to specific cellular receptor, which mediate their cellular uptake through endocytosis. lysosomotropic agents such as chloroquine and nh4cl can inhibit toxin-mediated cytotoxicity, suggesting that the endosomal acidification is involved in toxin intracellular trafficking. to determine whether or not the cellular activity of the recombinant toxins also require endosomal acidification, ct cells were pre-incubated with nh4cl for min before the toxin exposure. while the exposure of ct cells to rtcda or rtcdb induced the glucosylation of rac pretreatment of cells with ammonium chloride completely blocked such an activity. disruption of tight junctions of intestinal epithelial cells by recombinant toxins it is known that tcda and tcdb alter the structure of intestinal epithelia by disrupting tight junctions. we investigated whether or not the recombinant toxins we generated have similar activities. human intestinal epithelial hct- cells were cultured in a transwell for to days until transepithelial resistance reached Ω*cm and tight junctions were formed. rtcda did not alter cell-cell connections of hct- cells within hr of treatment but disrupted tight junctions after hr of treatment. rtcdb, at the same concentration with rtcda, completely disrupted the tight junctions of hct- within hr of treatment. discussion cdad is largely attributed to the bacterial exotoxins tcda and tcdb, although other virulence factors probably also play some roles. both toxins glucosylate rho gtpase family proteins, leading to their inactivation. to study the pathogenesis of cdad and host immune response to the toxins, and to use the toxins as a research tool for signal transduction involving rho gtpases, it is necessary to obtain large quantities of highly purified active tcda and tcdb. native toxins can be purified from toxigenic c. difficile culture supernatant. the purification process however usually involves multiple steps such as ultrafiltration, ammonium sulfate precipitation, and ion-exchange chromatography. in the case of tcda, affinity purification through thyroglobulin is available and relatively pure tcda can be obtained. tcdb however is often contaminated with some unknown substance even after extensive purification steps. gene engineering to generate recombinant toxins seems to be a logical approach for obtaining large quantities of pure toxin proteins. the e. coli system is popular and is widely used for the expression of recombinant proteins. several studies have reported to have cloned and expressed active c. difficile toxins in e. coli, but failed to demonstrate that the purified toxins were obtained from bacterial extracts. burger et al was the first to report a successful expression of recombinant tcda using bacillus megaterium system. recombinant tcdb, however, has not yet been stably expressed. in this study, we were successfully expressing a full-length recombinant tcdb in b. megaterium and greatly enhanced the expression level of recombinant tcda. because e. coli expression system is widely used and easy-to-handle, we have utilized various e. coli expression vector systems to clone and express both toxins and found that both recombinant tcda and tcdb were expressed in e. coli but we were unable to obtain the purified toxins from the bacterial lysate. the large size of the heterologous proteins may possibly be prone to protease digestion, and intrinsic protease activities of these toxins may also contribute to the instability. another possible reason for the inability to obtain purified recombinant toxins from e. coli is that the expression level was too low. in fact, large proteins are difficult to express at high levels in e. coli. furthermore, an unusually high content of at in these clostridial gene sequences and a different codon -usage make them particularly difficult to express at a high level in e. coli. there are several advantages associated with b. megaterium expression system including lack of alkaline phosphatase and endotoxin lps. more importantly, b. megaterium has been successfully used to express rtcda, we therefore decided to use this system. initially, we followed the reported cloning strategy but we were unsuccessful. this might be due to the extremely low ligation efficiency when putting several fragments together during the cloning. we then changed our strategy to a direct pcr amplification of whole toxin genes. however, this strategy was associated with a potential risk that pcr might introduce mutations due to the large size of the genes. because of the large size of both genes, it was impossible to sequence all constructs in order to identify the correct clones. we therefore took a different approach, screening for recombinant protein expression and cytotoxicity. we screened as many colonies as possible after ligation and then transformed these constructed shuttle vectors into b. megaterium protoplasts. the resulting colonies were subjected to screening again by examining the cytotoxic activity in bacterial crude extracts. the specific expression of recombinant toxins from the positive colonies was then determined by western blot. the plasmids encoding tcda or tcdb respectively were finally subjected to dna sequencing and no single mutation was found in either of the toxin genes. the average yields of recombinant tcda and tcdb were about – mg/liter of bacterial culture, which were much higher than that of native toxins from c. difficile culture, and up to 20-fold higher than the amount of rtcda reported by burger et al. the fact that we used a different shuttle vector from that of burger et al might explain our higher yields of recombinant proteins. in addition, we have found out a twelve- to sixteen-hour period of induction with xylose yielded more recombinant proteins than five hours as suggested by the manufacturer. the other possible explanation was that the tcda gene was fused into orf of the vector in the construct reported by burger et al, resulting a modified n-terminus of recombinant tcda. this modification might affect the efficiency of protein expression and/or its stability. we inserted the tcda or tcdb gene into the bsrgi site and the protein translation directly started from toxin gene, resulting in recombinant toxins without any n-terminal modification. to facilitate purification, a his tag was fused to the c-terminus of both toxins. one-step of ni-affinity chromatography resulted in approximately 70–80% purity of recombinant toxins as determined by coomassie staining of sds-page. to achieve a greater purity, an additional step was introduced after ni-affinity chromatography. for rtcda, thyroglobulin affinity purification was used. rtcdb was further purified by an anion-exchange fractionation. these additional purification steps for rtcda and rtcdb, respectively, led to highly pure recombinant toxins. no additional band was seen on a coomassie-stained gel. in addition, there were no detectable tlr or tlr stimulants within the purified recombinant toxins as determined by sensitive bioassays. this is important because any use of the impure proteins to study host immune response or signaling transduction would possibly result in unintended consequences. unlike e. coli, a protein secretory pathway is present in b. megaterium. a 28-amino-acid signal peptide of b. megaterium extracellular esterase lipa can direct the transport of expressed heterologous proteins into medium and the sequence is removed by a protease after secretion. because of the nature of exotoxins of native toxins and relatively easy purification from supernatant without the need for sonication or other means of cell disruptions, we sought to express rtcdb extracellularly. after inserting the gene of the leader signaling peptide at the 5' of tcdb, we were able to generate active secretory rtcdb and coomassie staining of concentrated supernatant on a sds-page gel and western blot showed rtcdb band. however, for some unknown reasons, the expression level of rtcdb was significantly decreased when the leader sequence was introduced to the construct. in addition, we found the secretion was very inefficient and the majority of toxins were still trapped inside of the bacteria, since the total bacterial lysate contained the majority of toxin activity. this secretory pathway may not be efficient for such a large size of protein. the low amount of rtcdb in the medium might also be due to the inefficient refolding after their extracellular transport.  in this study, we have successfully expressed the full-length of recombinant c. difficile toxin a and b proteins in b. megaterium and greatly improved the yield of the recombinant toxins. we were able to obtain – mg of highly purified rtcda or rtcdb from one liter of bacterial culture. these recombinant toxins had similar biological activities with their native counterparts. we believe the recombinant toxin proteins will provide an invaluable tool for studying the pathogenesis of c. difficile associated disease and host immune response to toxins, as well as tools for studying the signaling transduction involving rho gtpase family proteins. 
the recombinant toxins were purified from bacterial crude extracts. approximately – mg of highly purified recombinant toxins were obtained from one liter of bacterial culture. the resulting rtcda and rtcdb had similar molecular masses to the native toxins, and their biological activities were found to be similar to their native counterparts after an extensive examination. due to the large size and poor stability of the proteins, the active recombinant tcda and tcdb have been difficult to produce. the constructs were transformed into b. megaterium protoplasts and the protein expression was controlled under a xylose promoter.
 major clostridium difficile virulence factors are the exotoxins tcda and tcdb. due to the large size and poor stability of the proteins, the active recombinant tcda and tcdb have been difficult to produce.  the toxin genes tcda and tcdb were amplified by pcr using chromosomal dna from a toxigenic strain as a template, and cloned into a shuttle vector phis the sequences of both tcda and tcdb genes in the vector have been verified by dna sequencing. the constructs were transformed into b. megaterium protoplasts and the protein expression was controlled under a xylose promoter. the recombinant toxins were purified from bacterial crude extracts. approximately – mg of highly purified recombinant toxins were obtained from one liter of bacterial culture. the resulting rtcda and rtcdb had similar molecular masses to the native toxins, and their biological activities were found to be similar to their native counterparts after an extensive examination.  we have generated the full length and active recombinant tcda and tcdb in bacillus megaterium. 