BACKGROUND
the ciliate tetrahymena thermophila is a small non pathogenic protozoan and one of the best characterized eukaryotic unicellular organisms.
it has been used as a model system for basic molecular-, cell- and developmental biology research for decades  <cit> .
furthermore, it has been shown that t. thermophila is able to grow fast to high cell densities in cheap medium in a common bioreactor infrastructure  <cit> .
the beneficial growth characteristics combined together with the sub-cellular machinery for performing eukaryotic post-translational protein modifications and the established genetic engineering technology makes t. thermophila a potential alternative to more established expression systems  <cit> .
it has been reported that this system is capable of expressing gpi linked surface proteins from the parasitic ciliate ichthyophthirius multifiliis, the malaria parasite plasmodium falciparum and avian influenza virus antigen h5n <dig>  suggesting that t. thermophila could play an important role in vaccine development  <cit> .
moreover, we could show that t. thermophila is also able to express and secrete the functional human enzyme dnase i  <cit> .
the major challenge now is the transition of the tetrahymena based expression system from laboratory model to useful production platform.
it is therefore important to generate improved t. thermophila strains for maximizing the production yield and for meeting the specific requirements of a recombinant protein for a particular application.
this can be achieved by studying the processes that regulate synthesis, post-translational modification and sorting of recombinant proteins and by understanding how they can be controlled.
a further objective is the establishment of optimized fermentation procedures.
to date, upstream efforts have focused primarily on the improvement of growth conditions  <cit> .
little, if any, attention has been paid to determine cultivation parameters that enhance the productivity of recombinant proteins.
the availability of a robust reporter enzyme that allows to evaluate the efficiency of protein targeting on the one hand and to define optimal upstream processes on the other hand would be very benefical to attain this goal.
here we report the expression and secretion of the human intestinal alkaline phosphatase that can be used as a tool for the development of a high-yield glycoprotein expression system.
so far several alkaline phosphatases  of mammalian cells have been characterized.
these enzymes are divided into tissue-specific  and tissue non-specific  alkaline phosphatases  <cit> .
the hiap precursor protein consists of  <dig> amino acids , including a signal peptide at its n-terminus  and a gpi anchor signal  at its c-terminus.
the mature and processed enzyme lacking the first  <dig> and last  <dig> amino acids is localized on the cell surface due to a post-translational added gpi anchor.
it is postulated that the mature protein contains three potential n-glycosylation sites and two disulfide bridges   <cit> .
we prepared two constructs encoding the precursor hiap with and without a gpi anchor signal.
in order to avoid problems in expression due to the unique codon bias of ciliates a synthetic, codon-adapted gene, in which all of the very rare codons were replaced by highly frequented codons, was used.
we demonstrated that both variants could be expressed as functional and glycosylated proteins.
furthermore, we used the c-terminal truncated form of human intestinal alkaline phosphatase that lacks the gpi anchor signal in first optimization experiments.
RESULTS
expression and localization of human intestinal alkaline phosphatase in t. thermophila
in order to express the full-length precursor of hiap in ciliates we used a codon adapted artificial gene, according to the codon bias for highly expressed genes in t. thermophila  <cit> .
the complete hiap cdna was cloned into the rdna ori based ph4t <dig> vector  <cit> .
the expression of the hiap was regulated by the mtt1-promoter of t. thermophila  <cit> .
this promoter system can be induced by the addition of cadmium ions to the culture medium .
we transformed conjugating t. thermophila cells.
clones that were resistant against paromomycin were analyzed further.
cadmium was added to putative positive clones to induce hiap expression and the cell extracts were analyzed by western blot using a specific antiserum from sheep  originally derived against an internal epitope of human placental and intestinal alkaline phosphatase.
the non-induced cells of these clones and a wild type t. thermophila strain served as negative controls.
cell extracts of transformed cho cells containing the full-length hiap were used as positive control.
figure  <dig> shows that the antibody specifically recognizes one single protein with the apparental molecular weight of ~ <dig> kda, indicating that recombinant hiap was expressed by the transformed and cadmium treated ciliates.
hiap has three putative n-glycosylation sites at position, n <dig>  n <dig> and n <dig>  to analyze whether or not recombinant hiap from t. thermophila is glycosylated during its passage through the er and golgi membranes we performed a de-glycosylation assay, by using pngase f .
this enzyme is an amidase that cleaves between the amino acid asparagine  and the innermost n-acetylglucosamine residue  of high-mannose, hybrid and complex oligo-saccharides from n-linked glycoproteins.
we observed a significant reduction of the molecular weight of recombinant hiap from cho cells  as well as of hiap derived from ciliates in samples that were treated with pngase f .
these results indicate that n-glycans were present in both samples.
the elimination of the n-glycan structure of hiap derived from t. thermophila leads to a smaller band shift than the de-glycosylation of the protein expressed by cho cells.
this might result from a distinct bias of site occupancy, but previous data showed that the band shift difference was referable to a differential n-glycosylation pattern.
n-glycan moieties in t. thermophila were almost completely of the oligo-mannose type  <cit> , while the oligosaccharide structure of proteins from cho cells is of the hybrid or complex structure.
the de-glycosylated form of recombinant hiap derived from cho and ciliates had the same molecular weight, suggesting that the recombinant hiap enzyme is properly expressed.
to confirm the expression of a functional enzyme we prepared crude cell extracts of induced, non-induced and wild type cells and tested them for alkaline phosphatase activity.
the enzyme activity in extracts of cadmium induced cells was compared to the activity of extracts from non-induced and wild type cells.
we observed elevated phosphatase activity in the cells that were also positive in the western blot analysis, suggesting that hiap becomes expressed as an active, correctly folded enzyme.
in human tissues alkaline phosphatases are membrane associated by c-terminal gpi anchor moieties and localized on the cell surface  <cit> .
to monitor the functionality of the human gpi anchor signal in the ciliate t. thermophila, we analyzed the localization of recombinant hiap in ciliates by two independent approaches.
first, we made a simple fractionation experiment.
extracts from cadmium-induced cells were lyzed using a buffer without detergent  and centrifuged at  <dig>  × g. the basic cell extract , the supernatant  and the pellet fraction  were analyzed by sds-page and western blot analysis.
figure 3a illustrates that almost all of the recombinant hiap was found in the p <dig> fraction, suggesting that the recombinant hiap is membrane associated and not a soluble protein.
secondly, by using confocal microscopic analysis, we were able to confirm these data.
the top panel of figure 3b shows the normasky images and the bottom the immuno staining of tetrahymena cells.
the images show that the antibody l- <dig> specifically recognized a protein in only those clones in which the expression was induced by the addition of cadmium .
the wild type cells  and the non-induced cells  did not show signals above background.
surprisingly, the staining shows a distinct pattern and no continuous staining.
this pattern is shown in figure 3c at higher magnification.
a more detailed scanning analysis using  <dig>  μm scanning steps is shown in additional file  <dig>  figure  <dig> and additional file  <dig>  figure  <dig>  these figures clearly illustrate that recombinant hiap predominantly localizes to the surface of the cells.
additional structures stained within the cell probably correspond to transport vesicles and/or to intracellular membranous structures that contain hiap precursor proteins.
the previously shown localization of the heterologous expressed surface protein csp- <dig> a gpi anchored surface protein of plasmodium falciparum indirectly confirms this thesis because the immunofluorescence pattern of this protein shows significant similarities to hiap localization  <cit> .
these results suggest that the human full-length hiap protein is correctly processed and targeted in the ciliate system.
secretion of functional hiap into the medium
in the next experiments we used an expression cassette encoding a hiap truncation mutant that lacks the human gpi anchor signal to address two points.
firstly, we wanted to confirm that the endogenous human gpi anchor signal is responsible for the membrane attachment found in recombinant t. thermophila strains .
secondly, we wanted to investigate if hiap protein lacking the gpi anchor signal retains activity.
therefore, we analyzed aliquots of the supernatants after inducing hiap expression.
significant signals in western blot analysis were confirmed by phosphatase activity assays.
in comparison to the non-induced cells and the wild type control that did not show any alkaline phosphatase activity, we found a significant alkaline phosphatase activity in supernatants of cultures in which the expression of hiap was induced by the addition of cadmium.
as we did not find any endogenous alkaline phosphatase activity in the supernatants the measured intracellular activity in the wild type and non-induced cells is most probably due to a non-secreted, endogenous cytosolic alkaline phosphatase .
in conclusion, these data confirm that the gpi anchor signal is responsible for the membrane association of the heterologous expressed full-length hiap but seems to have little if any influence on its activity.
therefore, hiap seems to be an ideal reporter enzyme that will allow to analyze protein sorting and secretion in the t. thermophila expression system in future.
optimization and monitoring of secretion in t. thermophila
furthermore, the truncated form of hiap can be used as a technical tool, because it allows to quantify the influence of different fermentation parameters on the yield of expression and secretion.
for this purpose, we performed several optimization experiments and tested in how far parameters, like additional medium feeding and multiple cadmium inductions, have an influence on the yield of secreted recombinant hiap.
we performed several independent fermentations using a clone that secretes functional hiap without the gpi anchor signal .
three independent cultivations were done in parallel to test if multiple inductions with cadmium have an influence on the expression/secretion level.
unexpectedly, we found that multiple cadmium inductions did not lead to a further increase of hiap activity in the supernatant .
in contrast to multiple cadmium inductions continuous feeding with concentrated spp medium during the fermentation process resulted in significantly higher yields of extracellular hiap .
after  <dig> h  or  <dig> h  the extracellular hiap activity reached a maximum level and did not increase any further.
we wanted to test if the observed limited time period of hiap activity increase is due to a reduced secretion activity of the cells.
therefore, we performed a fermentation to measure the beta-glucosidase activity.
this enzyme activity usually increases during the late-log and the early-stationary phases  <cit> .
we found the highest levels of beta-glucosidase activity in the last  <dig> hours of the fermentation, indicating that the cells are viable and not inhibited in general secretion.
discussion
here we report the expression of human alkaline phosphatase by the ciliate t. thermophila.
a codon-adapted artificial gene encoding the full-length precursor enzyme is correctly translated into the functional hiap enzyme.
the heterologous expressed enzyme exhibited phosphatase activity and became n-glycosylated during its passage through the er/golgi compartments, suggesting that both, the human signal peptide as well as the human gpi anchor signal, were correctly recognized by the t. thermophila protein biosynthesis machinery.
furthermore, the expression cassette encoding the human full-length protein, including the c-terminal gpi anchor showed a membrane associated localization on the surface of the ciliate cells, whereas the c-terminal truncated protein, lacking this gpi anchor recognition signal was secreted into the medium.
the distinct localization pattern of the stained cells  raises the interesting question which signals determine protein targeting and localization within the ciliates' membranes.
previous results from the tiedtke group  showed that hydrolases of the phagolysosomal compartments  are secreted into the surrounding medium  <cit> .
tetrahymena possesses three secretory pathways, namely regulated exocytosis of dense-core granules, constitutive secretion of lysosomal enzymes and phagocytosis  <cit> .
at least, the first two of these routings can be used for recombinant protein secretion  <cit> .
however, the exact sites of secretion remain to be determined and therefore it is not really known whether or not different secretory and/or surface proteins use different exocytotic pathways.
in previous experiments we could show that fusion proteins of the t. thermophila pla <dig> signal peptide with the human dnase i became secreted and correctly processed  <cit> .
interestingly, the results we show here clearly indicate that also human signal peptides are sufficient for proper secretion.
as recently demonstrated by the turkewitz group  t. thermophila provides many excellent features as a model system to analyze regulated secretion in more detail  <cit> .
the truncated hiap used in this study provides an additional instrument to address such questions, because truncated hiap fused to signal peptides or to parts of proteins that are secreted continuously  or in a stimulus dependent manner  might allow deeper insights into the secretory pathways and their regulation.
illustrating the underlying factors that regulate expression and secretion is of paricular importance for high yield recombinant protein expression, because the efficiency of such processes correlates to the yield of biologically active protein.
however, scientific studies that focus on basic research rather than on biotechnological aspects will be necessary to elucidate these questions.
we performed fermentation experiments and used the truncated "hiap without gpi" system to monitor several modifications of the fermentation process.
we found that extracellular hiap activity increased only in the first  <dig> to  <dig> h after induction of the mtt <dig> promoter.
of course on the one hand this observation emphasizes that truncated hiap provides a powerful tool to quantify the secretion efficiency in living ciliate cells by a very sensitive assay.
but on the other hand this result raises the question why the hiap activity reached a stable level although the t. thermophila cells grew further to titers of  <dig>  ×  <dig> cells/ml.
one reasonable explanation might be that recombinant hiap is probably increasingly degraded by the ciliates' proteases of the cathepsin l family.
this is in agreement with studies that demonstrated that several endogenous hydrolases including proteases accumulate during the late stages in continuous fermentation processes  <cit> .
however, the hiap activity remained stable upon incubation in cell free medium from late log cultures for at least six hours .
an additional explanation is probably the induction characteristic of the applied mtt <dig> promoter.
the promoter of the metallothionein gene mtt <dig> of t. thermophila that was first introduced in  <dig> can be rapidly activated by simply adding cadmium ions to the growth medium  <cit> .
shang et al.
stated that mtt <dig> mrna reaches a maximum level at about  <dig> min after cadmium addition to the culture medium  <cit> .
however, in the same study it was shown that best expression levels of a recombinant antigen which expression was driven by the mtt <dig> promoter were detectable after  <dig> h of cadmium induction.
this observation fits to the finding that highest hiap expression level lagged behind the published data regarding endogenous mtt <dig> mrna level and that protein yield decreases after a certain time of induction.
multiple addition of cadmium did not lead to a "pseudo-constitutive" promoter activity in the cultures, suggesting that the inducible effect disappears the longer the fermentation lasts.
furthermore, it has been shown in t. pyriformis that a substantial portion of the added cadmium could accumulate inside the cells by chelating metallothioneines  <cit> .
a combination of both effects, protease degradation as well as the adaptation to the cadmium stress is also possible.
interestingly, the recombinant expression of human alkaline phosphatases shows a further fascinating aspect.
poelstra et al.
and bentala et al.
could show that human alkaline phosphatases are able to reduce mortality in mice infected with gram-negative bacteria  <cit> .
additionally, heemskerk et al.
were able to demonstrate in a phase iia study that alkaline phosphatase treatment improved renal function in severe sepsis or septic shock patients  <cit> .
these studies show that alkaline phosphatases, usually catalyzing the hydrolysis of monoesters with release of inorganic phosphate, are also capable of reducing toxicity of endotoxins/lipopolysaccharides , components of the outer membrane of gram-negative bacteria.
so far only few tools are available to fight against the clinic features of sepsis and the currently available therapies focused on antagonizing the most pro-inflammatory cytokines or by inactivation of the lps cascade for example by neutralizing anti lps antibodies  <cit> .
unfortunately, the studies also revealed that high dosage of alkaline phosphatases are necessary to attenuate lps toxicity and reduce mortality.
as human sources for alkaline phosphatases are limited, different approaches are tried to obtain sufficient amounts of this enzyme to proceed with the clinical tests.
ciliates that secrete large amounts of recombinant human phosphatases might provide a solution for this problem.
a general comparison of tetrahymena with common expression systems is given in additional file  <dig>
nam, ermonval and sharfstein  demonstrated production levels of  <dig> u/liter hiap with cho cells in a fed-batch bioreactor with a fermentation time of  <dig> to  <dig> days  <cit> .
later, nam et al.
produced  <dig> -  <dig>  u/liter with cho cells in a fed-batch bioreactor within  <dig> days  <cit> .
furthermore, chen, chang and chang  described a  <dig> liter fermentation of the yeast pichia pastoris with a duration time of at least  <dig> days and measured  <dig>  units/liter hiap in the supernatant  <cit> .
we could demonstrate here that up to  <dig>  u/liter hiap could be produced within  <dig> days fermentation time in a bench top bioreactor.
moreover, it has to be considered that typical steps for a process optimization, like the improvement of the yield by strain optimization and the development of an elaborated fermentation process, has not been undertaken so far.
thus, it should be possible to increase the production of hiap significantly.
compared to the production methods which have been described in the past, the here used tetrahymena based expression system seems to be an attractive alternative for the production of hiap.
CONCLUSIONS
the precursor of the human intestinal alkaline phosphatase becomes correctly processed and is expressed as a functional enzyme.
these data confirm the high potential of the ciliate based expression system  which offers the chance to develop a biopharmaceutical enzyme used against life-threatening sepsis caused by gram-negative bacteria.
furthermore, secreted hiap can be used as an instrument for scientific purposes as well as a reporter enzyme allowing the quantification of measures that are planned to optimize the ciliate expression system.

 the ciliate tetrahymena thermophila is a small non pathogenic protozoan and one of the best characterized eukaryotic unicellular organisms. it has been used as a model system for basic molecular-, cell- and developmental biology research for decades. furthermore, it has been shown that t. thermophila is able to grow fast to high cell densities in cheap medium in a common bioreactor infrastructure. the beneficial growth characteristics combined together with the sub-cellular machinery for performing eukaryotic post-translational protein modifications and the established genetic engineering technology makes t. thermophila a potential alternative to more established expression systems. it has been reported that this system is capable of expressing gpi linked surface proteins from the parasitic ciliate ichthyophthirius multifiliis, the malaria parasite plasmodium falciparum and avian influenza virus antigen h5n suggesting that t. thermophila could play an important role in vaccine development. moreover, we could show that t. thermophila is also able to express and secrete the functional human enzyme dnase i. the major challenge now is the transition of the tetrahymena based expression system from laboratory model to useful production platform. it is therefore important to generate improved t. thermophila strains for maximizing the production yield and for meeting the specific requirements of a recombinant protein for a particular application. this can be achieved by studying the processes that regulate synthesis, post-translational modification and sorting of recombinant proteins and by understanding how they can be controlled. a further objective is the establishment of optimized fermentation procedures. to date, upstream efforts have focused primarily on the improvement of growth conditions. little, if any, attention has been paid to determine cultivation parameters that enhance the productivity of recombinant proteins. the availability of a robust reporter enzyme that allows to evaluate the efficiency of protein targeting on the one hand and to define optimal upstream processes on the other hand would be very benefical to attain this goal. here we report the expression and secretion of the human intestinal alkaline phosphatase that can be used as a tool for the development of a high-yield glycoprotein expression system. so far several alkaline phosphatases of mammalian cells have been characterized. these enzymes are divided into tissue-specific and tissue non-specific alkaline phosphatases. the hiap precursor protein consists of amino acids, including a signal peptide at its n-terminus and a gpi anchor signal at its c-terminus. the mature and processed enzyme lacking the first and last amino acids is localized on the cell surface due to a post-translational added gpi anchor. it is postulated that the mature protein contains three potential n-glycosylation sites and two disulfide bridges. we prepared two constructs encoding the precursor hiap with and without a gpi anchor signal. in order to avoid problems in expression due to the unique codon bias of ciliates a synthetic, codon-adapted gene, in which all of the very rare codons were replaced by highly frequented codons, was used. we demonstrated that both variants could be expressed as functional and glycosylated proteins. furthermore, we used the c-terminal truncated form of human intestinal alkaline phosphatase that lacks the gpi anchor signal in first optimization experiments.  expression and localization of human intestinal alkaline phosphatase in t. thermophila in order to express the full-length precursor of hiap in ciliates we used a codon adapted artificial gene, according to the codon bias for highly expressed genes in t. thermophila. the complete hiap cdna was cloned into the rdna ori based ph4t vector. the expression of the hiap was regulated by the mtt1-promoter of t. thermophila. this promoter system can be induced by the addition of cadmium ions to the culture medium. we transformed conjugating t. thermophila cells. clones that were resistant against paromomycin were analyzed further. cadmium was added to putative positive clones to induce hiap expression and the cell extracts were analyzed by western blot using a specific antiserum from sheep originally derived against an internal epitope of human placental and intestinal alkaline phosphatase. the non-induced cells of these clones and a wild type t. thermophila strain served as negative controls. cell extracts of transformed cho cells containing the full-length hiap were used as positive control. figure shows that the antibody specifically recognizes one single protein with the apparental molecular weight of ~ kda, indicating that recombinant hiap was expressed by the transformed and cadmium treated ciliates. hiap has three putative n-glycosylation sites at position, n n and n to analyze whether or not recombinant hiap from t. thermophila is glycosylated during its passage through the er and golgi membranes we performed a de-glycosylation assay, by using pngase f. this enzyme is an amidase that cleaves between the amino acid asparagine and the innermost n-acetylglucosamine residue of high-mannose, hybrid and complex oligo-saccharides from n-linked glycoproteins. we observed a significant reduction of the molecular weight of recombinant hiap from cho cells as well as of hiap derived from ciliates in samples that were treated with pngase f. these results indicate that n-glycans were present in both samples. the elimination of the n-glycan structure of hiap derived from t. thermophila leads to a smaller band shift than the de-glycosylation of the protein expressed by cho cells. this might result from a distinct bias of site occupancy, but previous data showed that the band shift difference was referable to a differential n-glycosylation pattern. n-glycan moieties in t. thermophila were almost completely of the oligo-mannose type, while the oligosaccharide structure of proteins from cho cells is of the hybrid or complex structure. the de-glycosylated form of recombinant hiap derived from cho and ciliates had the same molecular weight, suggesting that the recombinant hiap enzyme is properly expressed. to confirm the expression of a functional enzyme we prepared crude cell extracts of induced, non-induced and wild type cells and tested them for alkaline phosphatase activity. the enzyme activity in extracts of cadmium induced cells was compared to the activity of extracts from non-induced and wild type cells. we observed elevated phosphatase activity in the cells that were also positive in the western blot analysis, suggesting that hiap becomes expressed as an active, correctly folded enzyme. in human tissues alkaline phosphatases are membrane associated by c-terminal gpi anchor moieties and localized on the cell surface. to monitor the functionality of the human gpi anchor signal in the ciliate t. thermophila, we analyzed the localization of recombinant hiap in ciliates by two independent approaches. first, we made a simple fractionation experiment. extracts from cadmium-induced cells were lyzed using a buffer without detergent and centrifuged at × g. the basic cell extract, the supernatant and the pellet fraction were analyzed by sds-page and western blot analysis. figure 3a illustrates that almost all of the recombinant hiap was found in the p fraction, suggesting that the recombinant hiap is membrane associated and not a soluble protein. secondly, by using confocal microscopic analysis, we were able to confirm these data. the top panel of figure 3b shows the normasky images and the bottom the immuno staining of tetrahymena cells. the images show that the antibody l- specifically recognized a protein in only those clones in which the expression was induced by the addition of cadmium. the wild type cells and the non-induced cells did not show signals above background. surprisingly, the staining shows a distinct pattern and no continuous staining. this pattern is shown in figure 3c at higher magnification. a more detailed scanning analysis using μm scanning steps is shown in additional file figure and additional file figure these figures clearly illustrate that recombinant hiap predominantly localizes to the surface of the cells. additional structures stained within the cell probably correspond to transport vesicles and/or to intracellular membranous structures that contain hiap precursor proteins. the previously shown localization of the heterologous expressed surface protein csp- a gpi anchored surface protein of plasmodium falciparum indirectly confirms this thesis because the immunofluorescence pattern of this protein shows significant similarities to hiap localization. these results suggest that the human full-length hiap protein is correctly processed and targeted in the ciliate system. secretion of functional hiap into the medium in the next experiments we used an expression cassette encoding a hiap truncation mutant that lacks the human gpi anchor signal to address two points. firstly, we wanted to confirm that the endogenous human gpi anchor signal is responsible for the membrane attachment found in recombinant t. thermophila strains. secondly, we wanted to investigate if hiap protein lacking the gpi anchor signal retains activity. therefore, we analyzed aliquots of the supernatants after inducing hiap expression. significant signals in western blot analysis were confirmed by phosphatase activity assays. in comparison to the non-induced cells and the wild type control that did not show any alkaline phosphatase activity, we found a significant alkaline phosphatase activity in supernatants of cultures in which the expression of hiap was induced by the addition of cadmium. as we did not find any endogenous alkaline phosphatase activity in the supernatants the measured intracellular activity in the wild type and non-induced cells is most probably due to a non-secreted, endogenous cytosolic alkaline phosphatase. in conclusion, these data confirm that the gpi anchor signal is responsible for the membrane association of the heterologous expressed full-length hiap but seems to have little if any influence on its activity. therefore, hiap seems to be an ideal reporter enzyme that will allow to analyze protein sorting and secretion in the t. thermophila expression system in future. optimization and monitoring of secretion in t. thermophila furthermore, the truncated form of hiap can be used as a technical tool, because it allows to quantify the influence of different fermentation parameters on the yield of expression and secretion. for this purpose, we performed several optimization experiments and tested in how far parameters, like additional medium feeding and multiple cadmium inductions, have an influence on the yield of secreted recombinant hiap. we performed several independent fermentations using a clone that secretes functional hiap without the gpi anchor signal. three independent cultivations were done in parallel to test if multiple inductions with cadmium have an influence on the expression/secretion level. unexpectedly, we found that multiple cadmium inductions did not lead to a further increase of hiap activity in the supernatant. in contrast to multiple cadmium inductions continuous feeding with concentrated spp medium during the fermentation process resulted in significantly higher yields of extracellular hiap. after h or h the extracellular hiap activity reached a maximum level and did not increase any further. we wanted to test if the observed limited time period of hiap activity increase is due to a reduced secretion activity of the cells. therefore, we performed a fermentation to measure the beta-glucosidase activity. this enzyme activity usually increases during the late-log and the early-stationary phases. we found the highest levels of beta-glucosidase activity in the last hours of the fermentation, indicating that the cells are viable and not inhibited in general secretion. discussion here we report the expression of human alkaline phosphatase by the ciliate t. thermophila. a codon-adapted artificial gene encoding the full-length precursor enzyme is correctly translated into the functional hiap enzyme. the heterologous expressed enzyme exhibited phosphatase activity and became n-glycosylated during its passage through the er/golgi compartments, suggesting that both, the human signal peptide as well as the human gpi anchor signal, were correctly recognized by the t. thermophila protein biosynthesis machinery. furthermore, the expression cassette encoding the human full-length protein, including the c-terminal gpi anchor showed a membrane associated localization on the surface of the ciliate cells, whereas the c-terminal truncated protein, lacking this gpi anchor recognition signal was secreted into the medium. the distinct localization pattern of the stained cells raises the interesting question which signals determine protein targeting and localization within the ciliates' membranes. previous results from the tiedtke group showed that hydrolases of the phagolysosomal compartments are secreted into the surrounding medium. tetrahymena possesses three secretory pathways, namely regulated exocytosis of dense-core granules, constitutive secretion of lysosomal enzymes and phagocytosis. at least, the first two of these routings can be used for recombinant protein secretion. however, the exact sites of secretion remain to be determined and therefore it is not really known whether or not different secretory and/or surface proteins use different exocytotic pathways. in previous experiments we could show that fusion proteins of the t. thermophila pla signal peptide with the human dnase i became secreted and correctly processed. interestingly, the results we show here clearly indicate that also human signal peptides are sufficient for proper secretion. as recently demonstrated by the turkewitz group t. thermophila provides many excellent features as a model system to analyze regulated secretion in more detail. the truncated hiap used in this study provides an additional instrument to address such questions, because truncated hiap fused to signal peptides or to parts of proteins that are secreted continuously or in a stimulus dependent manner might allow deeper insights into the secretory pathways and their regulation. illustrating the underlying factors that regulate expression and secretion is of paricular importance for high yield recombinant protein expression, because the efficiency of such processes correlates to the yield of biologically active protein. however, scientific studies that focus on basic research rather than on biotechnological aspects will be necessary to elucidate these questions. we performed fermentation experiments and used the truncated "hiap without gpi" system to monitor several modifications of the fermentation process. we found that extracellular hiap activity increased only in the first to h after induction of the mtt promoter. of course on the one hand this observation emphasizes that truncated hiap provides a powerful tool to quantify the secretion efficiency in living ciliate cells by a very sensitive assay. but on the other hand this result raises the question why the hiap activity reached a stable level although the t. thermophila cells grew further to titers of × cells/ml. one reasonable explanation might be that recombinant hiap is probably increasingly degraded by the ciliates' proteases of the cathepsin l family. this is in agreement with studies that demonstrated that several endogenous hydrolases including proteases accumulate during the late stages in continuous fermentation processes. however, the hiap activity remained stable upon incubation in cell free medium from late log cultures for at least six hours. an additional explanation is probably the induction characteristic of the applied mtt promoter. the promoter of the metallothionein gene mtt of t. thermophila that was first introduced in can be rapidly activated by simply adding cadmium ions to the growth medium. shang et al. stated that mtt mrna reaches a maximum level at about min after cadmium addition to the culture medium. however, in the same study it was shown that best expression levels of a recombinant antigen which expression was driven by the mtt promoter were detectable after h of cadmium induction. this observation fits to the finding that highest hiap expression level lagged behind the published data regarding endogenous mtt mrna level and that protein yield decreases after a certain time of induction. multiple addition of cadmium did not lead to a "pseudo-constitutive" promoter activity in the cultures, suggesting that the inducible effect disappears the longer the fermentation lasts. furthermore, it has been shown in t. pyriformis that a substantial portion of the added cadmium could accumulate inside the cells by chelating metallothioneines. a combination of both effects, protease degradation as well as the adaptation to the cadmium stress is also possible. interestingly, the recombinant expression of human alkaline phosphatases shows a further fascinating aspect. poelstra et al. and bentala et al. could show that human alkaline phosphatases are able to reduce mortality in mice infected with gram-negative bacteria. additionally, heemskerk et al. were able to demonstrate in a phase iia study that alkaline phosphatase treatment improved renal function in severe sepsis or septic shock patients. these studies show that alkaline phosphatases, usually catalyzing the hydrolysis of monoesters with release of inorganic phosphate, are also capable of reducing toxicity of endotoxins/lipopolysaccharides, components of the outer membrane of gram-negative bacteria. so far only few tools are available to fight against the clinic features of sepsis and the currently available therapies focused on antagonizing the most pro-inflammatory cytokines or by inactivation of the lps cascade for example by neutralizing anti lps antibodies. unfortunately, the studies also revealed that high dosage of alkaline phosphatases are necessary to attenuate lps toxicity and reduce mortality. as human sources for alkaline phosphatases are limited, different approaches are tried to obtain sufficient amounts of this enzyme to proceed with the clinical tests. ciliates that secrete large amounts of recombinant human phosphatases might provide a solution for this problem. a general comparison of tetrahymena with common expression systems is given in additional file nam, ermonval and sharfstein demonstrated production levels of u/liter hiap with cho cells in a fed-batch bioreactor with a fermentation time of to days. later, nam et al. produced - u/liter with cho cells in a fed-batch bioreactor within days. furthermore, chen, chang and chang described a liter fermentation of the yeast pichia pastoris with a duration time of at least days and measured units/liter hiap in the supernatant. we could demonstrate here that up to u/liter hiap could be produced within days fermentation time in a bench top bioreactor. moreover, it has to be considered that typical steps for a process optimization, like the improvement of the yield by strain optimization and the development of an elaborated fermentation process, has not been undertaken so far. thus, it should be possible to increase the production of hiap significantly. compared to the production methods which have been described in the past, the here used tetrahymena based expression system seems to be an attractive alternative for the production of hiap.  the precursor of the human intestinal alkaline phosphatase becomes correctly processed and is expressed as a functional enzyme. these data confirm the high potential of the ciliate based expression system which offers the chance to develop a biopharmaceutical enzyme used against life-threatening sepsis caused by gram-negative bacteria. furthermore, secreted hiap can be used as an instrument for scientific purposes as well as a reporter enzyme allowing the quantification of measures that are planned to optimize the ciliate expression system. 
tetrahymena thermophila possesses many attributes that render it an attractive host for the expression of recombinant proteins. furthermore, it was demonstrated that the enzyme was modified with n-glycosylation and localized on the surface membrane by the c-terminal gpi anchor. furthermore, it has been demonstrated that t. thermophila is also able to produce a functional human dnase i. the present study investigates the heterologous expression of the functional human intestinal alkaline phosphatase using t. thermophila and thereby presents a powerful tool for the optimization of the ciliate-based expression system. functional and full length human intestinal alkaline phosphatase was expressed by t. thermophila using a codon-adapted gene containing the native signal-peptide and gpi anchor attachment signal. with the expression of hiap, not only a protein of commercial interest could be produced, but also a reporter enzyme that offers the possibility to analyze t. thermophila genes that play a role in the regulation of protein secretion. surface proteins from the parasites ichthyophthirius multifiliis and plasmodium falciparum and avian influenza virus antigen h5n were displayed on the cell membrane of this ciliate. hiap activity in the cell extract of transformants suggested that the hiap gene was successfully expressed. in a first approach to establish a high level expression system up to u/liter were produced in a time frame of two days, which exceeds the production rate of other published expression systems for this enzyme. additionally, the fact that ciliates do not secrete an endogenous alkaline phosphatase provides the possibility to use the truncated hiap as a reporter enzyme, allowing the quantification of measures that will be necessary for further optimization of the host strains and the fermentation processes.
 tetrahymena thermophila possesses many attributes that render it an attractive host for the expression of recombinant proteins. surface proteins from the parasites ichthyophthirius multifiliis and plasmodium falciparum and avian influenza virus antigen h5n were displayed on the cell membrane of this ciliate. furthermore, it has been demonstrated that t. thermophila is also able to produce a functional human dnase i. the present study investigates the heterologous expression of the functional human intestinal alkaline phosphatase using t. thermophila and thereby presents a powerful tool for the optimization of the ciliate-based expression system.  functional and full length human intestinal alkaline phosphatase was expressed by t. thermophila using a codon-adapted gene containing the native signal-peptide and gpi anchor attachment signal. hiap activity in the cell extract of transformants suggested that the hiap gene was successfully expressed. furthermore, it was demonstrated that the enzyme was modified with n-glycosylation and localized on the surface membrane by the c-terminal gpi anchor. a c-terminally truncated version of hiap lacking the gpi anchor signal peptide was secreted into the medium as an active enzyme. in a first approach to establish a high level expression system up to u/liter were produced in a time frame of two days, which exceeds the production rate of other published expression systems for this enzyme.  with the expression of hiap, not only a protein of commercial interest could be produced, but also a reporter enzyme that offers the possibility to analyze t. thermophila genes that play a role in the regulation of protein secretion. additionally, the fact that ciliates do not secrete an endogenous alkaline phosphatase provides the possibility to use the truncated hiap as a reporter enzyme, allowing the quantification of measures that will be necessary for further optimization of the host strains and the fermentation processes. 