BACKGROUND
the product range for recombinant biopharmaceuticals includes relatively simple proteins, for example insulin, and ends with candidates that require extensive secondary modification like erythropoietin or coagulation factor ix  <cit> .
commercial-scale production of proteins  nowadays is mainly performed with bacteria or mammalian cell-culture systems  <cit> .
bacterial production systems are highly efficient and well established for the production of comparably simple proteins but they come to their limits if complex posttranslational processing is required.
one major problem is the absence of protein glycosylation in prokaryotes.
mammalian cell lines, on the other hand, perform human-like posttranslational modifications and offer a high product quality but hold risks of product contamination and high overall production costs .
plants have emerged as a safe and cost-effective alternative to the traditional systems .
they synthesize proteins with correct assemblage and the posttranslational modifications of higher eukaryotes.
in contrast to microbial systems, glycosylated plant proteins carry complex-type glycans with the same core structure as human glycoproteins.
the glycosylation pattern of plant proteins, however, differs from that in humans concerning two residues linked to the core structure as well as terminal sugar moieties  <cit> .
the adaptation of the plant glycan pattern to its human counterpart  poses a challenge for the implementation of plant systems with regard to the production of recombinant pharmaceutical proteins  <cit>  as differences in the glycan structure could substantially alter protein characteristics and even act immunogenically in patients  <cit> .
the moss physcomitrella patens is the only known plant in which homologous recombination occurs in a frequency that allows its application as an engineering tool for targeted knock out and gene replacement.
with this technique a humanisation of the moss glycosylation pattern was performed recently  <cit> .
together with photoautotrophic growth in bioreactors these advantages make physcomitrella an ideal system for the production of plant-made pharmaceuticals  <cit> .
in molecular farming approaches, recombinant proteins, which require correct posttranslational modifications, are often targeted to the secretory pathway.
this, on the one hand, is due to the fact that enzymes responsible for various modifications, e. g. glycosylation and protein trimming, are located within the membranes of endoplasmic reticulum  and golgi apparatus.
on the other hand, secretion of the protein of interest into the culture medium separates it from the pool of intracellular proteins and therefore drastically facilitates its purification and reduces costs for downstream processing  <cit> .
a common way to achieve secretion of recombinant proteins is the utilisation of the native signal peptide, if any is present.
the mechanism of protein secretion depends on the recognition of an n-terminal signal peptide and is conserved among all eukaryotes  <cit> .
though the general hydrophobic character of secretion signals is conserved between plants and animals, they are not universally interchangeable  <cit> .
additional features might contribute to the efficiency of signal peptide recognition  <cit> .
in order to establish moss as a production system for recombinant proteins, we intended to enrich our toolboxes with a set of endogenous signal sequences from the moss.
the availability of physcomitrella secretion signals gives us the opportunity to secrete even proteins that are located within the cytosol in their host organism and thus lack a secretion signal.
here, we describe the comparison of two heterologous signal peptides from human origin, the vascular endothelial growth factor  and the coagulation factor ix , respectively, in the moss.
evaluation of a heterologous signal and the signal peptide of physcomitrella aspartic proteinase ppap <dig>  <cit>  revealed better secretion of recombinant hvegf  with the moss signal.
in addition, the secretion efficiency of five putative signal peptides originating from recently isolated and identified physcomitrella extracellular proteins xyloglucan endotransglycosylase/hydrolase , pectin methylesterase , fasciclin-like protein , carbonic anhydrase-like protein , and lipid-transfer protein   was tested in comparison to the ppap <dig> secretion signal.
these signal sequences were fused to hvegf and amounts of secreted rhvegf were quantified by elisa measurements.
all of the investigated moss signal peptides showed at least comparable or even higher secretion efficiency than the human signals.
besides, we underline the functionality of a transient production technique, that allows the fast production of recombinant proteins  <cit> .
within the field of molecular farming these techniques are essential for proof-of-concept studies prior to the establishment of production lines.
RESULTS
efficiency of human secretion signals in physcomitrella patens
in order to improve transient production and secretion of pharmaceutically interesting proteins in the moss physcomitrella patens, we first tested the efficiency of two human signal peptides.
these were derived from the human blood clotting factor ix  and the vascular endothelial growth factor .
the secretion signals of both proteins, fsp and vsp, respectively, were tested with hvegf as a reporter gene.
physcomitrella protoplasts were transiently transfected with the respective plasmids followed by quantification of extra- and intracellular concentrations of the recombinant proteins by elisa .
testing the hfix signal peptide fsp, the moss cells secreted on average  <dig>  ng/ml recombinant hvegf  to the culture supernatant, whereas the hvegf signal vsp yielded a slightly higher average value .
comparison with intracellular concentrations of rhvegf proved that both heterologous signals from human origin were quite effective in secreting the reporter protein from moss cells.
transient production of recombinant glutathione s-transferase
having proven the general functionality of heterologous secretion signals we aimed to compare the efficiency of a human and a moss signal peptide.
we chose vsp that secreted a slightly higher amount of the reporter protein, and asp, a moss signal sequence of the recently characterised aspartic proteinase ppap <dig>  <cit> .
the signal sequences were fused to the cdna of glutathione s-transferase  from schistosoma japonicum, which served as a reporter protein.
five days after transient transfection of moss cells, reporter concentrations were quantified by elisa .
with the moss signal peptide asp, the protoplasts secreted between  <dig>  and  <dig>  ng/ml rgst into the culture supernatant.
on the contrary, the human signal peptide yielded values in the range from  <dig>  to  <dig>  ng/ml.
taken the means of these values, when using the moss signal peptide, the concentration of rgst in the medium  was almost three times higher than with the human signal  .
for the quantification of intracellular rgst, the crude protein extracts were directly measured by elisa .
the relation of the values correlated to the extracellular situation.
asp-gst-transfected protoplasts contained  <dig>  ng/ml rgst whereas those transfected with vsp-gst reached a mean value of no more than  <dig>  ng/ml.
for visualisation of recombinant gst on western blots , gst was affinity-purified by using gsh-sepharose, and the eluted proteins were separated by non-reducing sds-page.
gst was detected with an anti-gst antibody.
the blots showed the main bands at the expected size of  <dig> kda for the gst protein.
the weak additional higher band was caused by dimer formation.
weak bands detected in the control lanes may derive from cross-reactivity of the anti-gst antibody to moss gst or be an artefact from the protein purification procedure.
transient production of recombinant human vegf
to examine whether the supremacy of the moss signal is specific for gst, we subsequently analysed the secretion efficiency of asp and vsp with the hvegf reporter protein.
when the natural signal peptide was used, the amount of extracellular rhvegf ranged between  <dig>  and  <dig>  ng/ml .
using the moss signal peptide resulted in a more than fivefold increase of rhvegf in the culture medium .
the differences in extracellular rhvegf accumulation of asp-vegf vs. vsp-vegf-transfected cells were also reflected in the western analysis .
additionally, the amounts of intracellular rhvegf were determined by elisa.
asp-vegf-transfected protoplasts contained  <dig>  ng/ml rhvegf.
those transfected with vsp-vegf reached an average value of  <dig>  ng/ml.
highly efficient secretion by endogenous signal peptides
comparison of the intracellularly retained and extracellular proportions of rgst and rhvegf, respectively, demonstrates that secretion from the moss cells was very efficient .
most of rgst, i.e.
<dig> %, was secreted to the culture medium.
up to  <dig> % of the produced rhvegf accumulated in the culture medium, whereas no significant amounts were found inside the cells, neither with the moss signal nor with the human one.
furthermore, the ratio of intra- to extracellular protein did not differ significantly between the human and the moss secretion signal.
with respect to the biological processes, we suggest as the most probable among several possible reasons for the difference in secretion efficiency a less efficient translation process when using the human signal .
after having proven the high efficiency of one endogenous moss signal peptide in the moss system, we aimed to study the efficiency of additional moss signal sequences and tested five other putative secretion signals from physcomitrella in transient transfection assays.
these signal peptides originated from putative extracellular proteins isolated from moss culture medium .
the signal sequences of ppflp , ppltp , pppme <dig> , ppxth <dig> , ppcalp  as well as ppap <dig> were fused to rhvegf in order to analyse their secretion efficiency by elisa after transient transfection of moss protoplasts.
all further investigated moss signal peptides showed comparable or even higher secretion efficiencies than the ppap <dig> signal asp .
the rhvegf amount in the medium achieved by the signal peptides of pppme <dig> and ppxth <dig> was approximately 80% higher than with the ppap <dig> signal.
a comparison of the different moss signal peptides is shown in fig.
<dig>  according to signalp, for the six cloned sequences the probabilities for being a signal peptide were always higher than 99% and their functionality in vivo was proven as demonstrated above.
all of the signals comprise many hydrophobic amino acid residues in their central part indicating the hydrophobic region.
the n-terminal part has at least one positive residue in most of the signals , whereas the c-terminal part always contains a small and neutral residue at position - <dig> and - <dig> with respect to the cleavage site as it is proposed for signal sequences  <cit> .
the probability for correct prediction of the cleavage site varied from  <dig> % for ppcalp to  <dig> % for ppflp.
high variations were also found regarding the overall length of the signal sequences, which ranges from  <dig> to  <dig> amino acids.
analysis of available physcomitrella sequences resulted in an average length of  <dig> amino acids for  <dig> signal peptides predicted.
taken together, the results of the transient production of recombinant proteins demonstrate that the secretion machinery of physcomitrella works very efficiently.
furthermore it was shown that the utilisation of an endogenous signal peptide can lead to a remarkable increase in recombinant protein production in comparison with the heterologous sorting signals used in this study.
discussion
an important issue in molecular farming is the accumulation and downstream processing of recombinant proteins.
mostly, the proteins are targeted to the secretory pathway as the complete set of enzymes responsible for posttranslational modifications, especially for protein glycosylation, is located within the secretory compartments, i.e.
er and golgi apparatus.
in case of using suspension cultures as a production system secretion of proteins provides the additional advantage of easier protein purification.
several systems involving secretion in protein production have been established.
among these are both, cell suspension cultures and whole plant systems, e.g.
rhizosecretion  <cit>  or production in the water plant lemna  <cit> .
seed storage of the recombinant product also involves secretory targeting, as deposition in protein storage vacuoles is achieved by traffic via the secretory pathway  <cit> .
a common practice for the production and secretion of human proteins in plants is to use their native signal peptide.
this is possible because of the conservation of the signal recognition- and er-insertion mechanisms throughout eukaryotes  <cit>  and circumvents laborious steps of signal engineering.
however, the amino-acid composition of signal peptides is extremely variable and the exact mechanism of their recognition by srp and the translocon is not yet completely understood.
their determining feature is the hydrophobicity of a ten to fifteen residue core sequence, which surprisingly does not show any sequence conservation  <cit> .
the functionality of the signals is only marginally impaired by amino-acid exchanges, as long as the hydrophobic core is not affected  <cit> .
exchanges within the core can in contrast totally impede signal recognition, even when leading to a higher hydrophobicity  <cit> .
a closer look at the nature of the six moss signal peptides analysed in this study revealed a high percentage of the hydrophobic amino acids l, f, i, m, v and w  <cit>  in the central part of the signal peptides, even though the hydrophobic stretches vary in length and position.
n-terminal of the hydrophobic centre positively charged residues have been proposed  <cit> .
except for ppcalp, all of the moss signal sequence n-termini contain at least one arginine residue.
for the signal peptide of ppcalp, which has no positively charged residue, the initiation methionine could perhaps compensate the lack of arginine  <cit> .
all of the investigated signal sequences follow the rule, which means that the two amino acids at position one and three previous to the cleavage site have to be small and neutral  <cit> .
the hmm cleavage site prediction from signalp showed a rather low probability for the signal sequences of pppme <dig>  and of ppcalp .
additionally, in case of ppcalp, the signalp-neural network predicted a signal peptide length of  <dig> amino acids instead of  <dig> amino acids which was predicted with signalp-hmm and used in our study.
however, experimental data showed that the sequences for both ppcalp and pppme <dig> were not only functional, but resulted in a very efficient secretion of the rhvegf protein.
in addition, cleavage site prediction of asp and vsp was verified experimentally.
the analysis revealed that secretion dropped drastically when the last amino acid of a correctly predicted signal was deleted .
the average length of eukaryotic signal peptides was calculated to be  <dig> amino acids  <cit> , whereas the average length of  <dig> analysed physcomitrella signal peptides is  <dig> residues.
although some correlations between the analysed physcomitrella signal peptides and other eukaryotic secretion signals can be found, the variations within the amino acid composition remain high  <cit> .
a functional role of additional, most likely structural determinants of signal peptides is assumed  <cit> .
previous studies evaluating endogenous vs. heterologous signal sequences in several species of the plant and animal kingdoms revealed for the majority a higher efficiency of kingdom-specific signals  <cit> .
as secretion is one of the bottlenecks when establishing a production system, we aimed to define highly efficient signals to transport the recombinant proteins to the culture medium of the moss bioreactor system  <cit> .
thus we decided to compare six endogenous moss signal peptides plus two heterologous sequences from human origin.
all of the moss signals turned out to drive secretion more efficiently than the human signal peptides.
two signals, the moss-derived asp and the human vsp, were analysed in more detail with two different reporters, rgst  and rhvegf , respectively.
by secretion of the recombinant reporter protein gst we could show a threefold increase in extracellular accumulation with the asp sequence compared to the human vsp.
with the moss secretion signal, extracellular rhvegf amount was up to fivefold more.
surprisingly, we did not find an increased intracellular accumulation in case of the human signal peptide.
amongst several possible alternatives , our preferred explanation is that recognition by the srp and maybe er-insertion of the human signal peptide is suboptimal thus decreasing the translation rate of the recombinant protein.
the differences in secretion efficiency clearly prove that besides hydrophobicity, some additional features, that may have a species-specific character, play a crucial role in signal recognition.
as it is known that srp occupies the ef-binding-site of the ribosome and thereby temporarily arrests translation  <cit> , the cause for a reduced production rate with the human signal could be found in a hindered dissociation of srp after docking of the translation complex to the translocon.
the second important interaction of the nascent signal peptide is performed with er-membrane lipids as well as the translocon itself  <cit> .
this interaction could demand unknown structural features of a signal peptide, which are not completely cross-host compatible, as mammalian and plant membranes differ significantly in their lipid composition  <cit> .
however, besides a translational inhibition caused by hindered srp-dissociation, alternative explanations for the worse performance of the human signals do exist: the protein in part may fail to be properly folded  and is subsequently degraded.
alternatively, transcription of that particular construct may be inefficient.
the only possible determinant for such events can be found in the relatively short signal peptide sequences themselves, as all used vectors do not differ in any other detail.
apart from the above-mentioned translational inhibition, additional influence of the signal peptide on recombinant protein stability could only take place during, or shortly after er-insertion.
when the translation of the protein is completed, the signal peptides are cleaved.
thereafter, recombinant products do not differ in any residue and can therefore not exhibit different stabilities.
regulatory, post-targeting functions of cleaved signals are proposed by other authors and can therefore not be completely excluded in our case  <cit> .
CONCLUSIONS
even if the structural background remains to be elucidated, our results demonstrate that the six moss sequences are more efficient than the two human ones used in our study in promoting secretion of recombinant human vegf.
in the interplay with optimised gene expression  <cit>  and product stabilisation  <cit> , the utilisation of moss-endogenous secretion signals could greatly enhance the productivity of the moss-bioreactor system.

 the product range for recombinant biopharmaceuticals includes relatively simple proteins, for example insulin, and ends with candidates that require extensive secondary modification like erythropoietin or coagulation factor ix. commercial-scale production of proteins nowadays is mainly performed with bacteria or mammalian cell-culture systems. bacterial production systems are highly efficient and well established for the production of comparably simple proteins but they come to their limits if complex posttranslational processing is required. one major problem is the absence of protein glycosylation in prokaryotes. mammalian cell lines, on the other hand, perform human-like posttranslational modifications and offer a high product quality but hold risks of product contamination and high overall production costs. plants have emerged as a safe and cost-effective alternative to the traditional systems. they synthesize proteins with correct assemblage and the posttranslational modifications of higher eukaryotes. in contrast to microbial systems, glycosylated plant proteins carry complex-type glycans with the same core structure as human glycoproteins. the glycosylation pattern of plant proteins, however, differs from that in humans concerning two residues linked to the core structure as well as terminal sugar moieties. the adaptation of the plant glycan pattern to its human counterpart poses a challenge for the implementation of plant systems with regard to the production of recombinant pharmaceutical proteins as differences in the glycan structure could substantially alter protein characteristics and even act immunogenically in patients. the moss physcomitrella patens is the only known plant in which homologous recombination occurs in a frequency that allows its application as an engineering tool for targeted knock out and gene replacement. with this technique a humanisation of the moss glycosylation pattern was performed recently. together with photoautotrophic growth in bioreactors these advantages make physcomitrella an ideal system for the production of plant-made pharmaceuticals. in molecular farming approaches, recombinant proteins, which require correct posttranslational modifications, are often targeted to the secretory pathway. this, on the one hand, is due to the fact that enzymes responsible for various modifications, e. g. glycosylation and protein trimming, are located within the membranes of endoplasmic reticulum and golgi apparatus. on the other hand, secretion of the protein of interest into the culture medium separates it from the pool of intracellular proteins and therefore drastically facilitates its purification and reduces costs for downstream processing. a common way to achieve secretion of recombinant proteins is the utilisation of the native signal peptide, if any is present. the mechanism of protein secretion depends on the recognition of an n-terminal signal peptide and is conserved among all eukaryotes. though the general hydrophobic character of secretion signals is conserved between plants and animals, they are not universally interchangeable. additional features might contribute to the efficiency of signal peptide recognition. in order to establish moss as a production system for recombinant proteins, we intended to enrich our toolboxes with a set of endogenous signal sequences from the moss. the availability of physcomitrella secretion signals gives us the opportunity to secrete even proteins that are located within the cytosol in their host organism and thus lack a secretion signal. here, we describe the comparison of two heterologous signal peptides from human origin, the vascular endothelial growth factor and the coagulation factor ix, respectively, in the moss. evaluation of a heterologous signal and the signal peptide of physcomitrella aspartic proteinase ppap revealed better secretion of recombinant hvegf with the moss signal. in addition, the secretion efficiency of five putative signal peptides originating from recently isolated and identified physcomitrella extracellular proteins xyloglucan endotransglycosylase/hydrolase, pectin methylesterase, fasciclin-like protein, carbonic anhydrase-like protein, and lipid-transfer protein was tested in comparison to the ppap secretion signal. these signal sequences were fused to hvegf and amounts of secreted rhvegf were quantified by elisa measurements. all of the investigated moss signal peptides showed at least comparable or even higher secretion efficiency than the human signals. besides, we underline the functionality of a transient production technique, that allows the fast production of recombinant proteins. within the field of molecular farming these techniques are essential for proof-of-concept studies prior to the establishment of production lines.  efficiency of human secretion signals in physcomitrella patens in order to improve transient production and secretion of pharmaceutically interesting proteins in the moss physcomitrella patens, we first tested the efficiency of two human signal peptides. these were derived from the human blood clotting factor ix and the vascular endothelial growth factor. the secretion signals of both proteins, fsp and vsp, respectively, were tested with hvegf as a reporter gene. physcomitrella protoplasts were transiently transfected with the respective plasmids followed by quantification of extra- and intracellular concentrations of the recombinant proteins by elisa. testing the hfix signal peptide fsp, the moss cells secreted on average ng/ml recombinant hvegf to the culture supernatant, whereas the hvegf signal vsp yielded a slightly higher average value. comparison with intracellular concentrations of rhvegf proved that both heterologous signals from human origin were quite effective in secreting the reporter protein from moss cells. transient production of recombinant glutathione s-transferase having proven the general functionality of heterologous secretion signals we aimed to compare the efficiency of a human and a moss signal peptide. we chose vsp that secreted a slightly higher amount of the reporter protein, and asp, a moss signal sequence of the recently characterised aspartic proteinase ppap. the signal sequences were fused to the cdna of glutathione s-transferase from schistosoma japonicum, which served as a reporter protein. five days after transient transfection of moss cells, reporter concentrations were quantified by elisa. with the moss signal peptide asp, the protoplasts secreted between and ng/ml rgst into the culture supernatant. on the contrary, the human signal peptide yielded values in the range from to ng/ml. taken the means of these values, when using the moss signal peptide, the concentration of rgst in the medium was almost three times higher than with the human signal. for the quantification of intracellular rgst, the crude protein extracts were directly measured by elisa. the relation of the values correlated to the extracellular situation. asp-gst-transfected protoplasts contained ng/ml rgst whereas those transfected with vsp-gst reached a mean value of no more than ng/ml. for visualisation of recombinant gst on western blots, gst was affinity-purified by using gsh-sepharose, and the eluted proteins were separated by non-reducing sds-page. gst was detected with an anti-gst antibody. the blots showed the main bands at the expected size of kda for the gst protein. the weak additional higher band was caused by dimer formation. weak bands detected in the control lanes may derive from cross-reactivity of the anti-gst antibody to moss gst or be an artefact from the protein purification procedure. transient production of recombinant human vegf to examine whether the supremacy of the moss signal is specific for gst, we subsequently analysed the secretion efficiency of asp and vsp with the hvegf reporter protein. when the natural signal peptide was used, the amount of extracellular rhvegf ranged between and ng/ml. using the moss signal peptide resulted in a more than fivefold increase of rhvegf in the culture medium. the differences in extracellular rhvegf accumulation of asp-vegf vs. vsp-vegf-transfected cells were also reflected in the western analysis. additionally, the amounts of intracellular rhvegf were determined by elisa. asp-vegf-transfected protoplasts contained ng/ml rhvegf. those transfected with vsp-vegf reached an average value of ng/ml. highly efficient secretion by endogenous signal peptides comparison of the intracellularly retained and extracellular proportions of rgst and rhvegf, respectively, demonstrates that secretion from the moss cells was very efficient. most of rgst, i.e. %, was secreted to the culture medium. up to % of the produced rhvegf accumulated in the culture medium, whereas no significant amounts were found inside the cells, neither with the moss signal nor with the human one. furthermore, the ratio of intra- to extracellular protein did not differ significantly between the human and the moss secretion signal. with respect to the biological processes, we suggest as the most probable among several possible reasons for the difference in secretion efficiency a less efficient translation process when using the human signal. after having proven the high efficiency of one endogenous moss signal peptide in the moss system, we aimed to study the efficiency of additional moss signal sequences and tested five other putative secretion signals from physcomitrella in transient transfection assays. these signal peptides originated from putative extracellular proteins isolated from moss culture medium. the signal sequences of ppflp, ppltp, pppme, ppxth, ppcalp as well as ppap were fused to rhvegf in order to analyse their secretion efficiency by elisa after transient transfection of moss protoplasts. all further investigated moss signal peptides showed comparable or even higher secretion efficiencies than the ppap signal asp. the rhvegf amount in the medium achieved by the signal peptides of pppme and ppxth was approximately 80% higher than with the ppap signal. a comparison of the different moss signal peptides is shown in fig. according to signalp, for the six cloned sequences the probabilities for being a signal peptide were always higher than 99% and their functionality in vivo was proven as demonstrated above. all of the signals comprise many hydrophobic amino acid residues in their central part indicating the hydrophobic region. the n-terminal part has at least one positive residue in most of the signals, whereas the c-terminal part always contains a small and neutral residue at position - and - with respect to the cleavage site as it is proposed for signal sequences. the probability for correct prediction of the cleavage site varied from % for ppcalp to % for ppflp. high variations were also found regarding the overall length of the signal sequences, which ranges from to amino acids. analysis of available physcomitrella sequences resulted in an average length of amino acids for signal peptides predicted. taken together, the results of the transient production of recombinant proteins demonstrate that the secretion machinery of physcomitrella works very efficiently. furthermore it was shown that the utilisation of an endogenous signal peptide can lead to a remarkable increase in recombinant protein production in comparison with the heterologous sorting signals used in this study. discussion an important issue in molecular farming is the accumulation and downstream processing of recombinant proteins. mostly, the proteins are targeted to the secretory pathway as the complete set of enzymes responsible for posttranslational modifications, especially for protein glycosylation, is located within the secretory compartments, i.e. er and golgi apparatus. in case of using suspension cultures as a production system secretion of proteins provides the additional advantage of easier protein purification. several systems involving secretion in protein production have been established. among these are both, cell suspension cultures and whole plant systems, e.g. rhizosecretion or production in the water plant lemna. seed storage of the recombinant product also involves secretory targeting, as deposition in protein storage vacuoles is achieved by traffic via the secretory pathway. a common practice for the production and secretion of human proteins in plants is to use their native signal peptide. this is possible because of the conservation of the signal recognition- and er-insertion mechanisms throughout eukaryotes and circumvents laborious steps of signal engineering. however, the amino-acid composition of signal peptides is extremely variable and the exact mechanism of their recognition by srp and the translocon is not yet completely understood. their determining feature is the hydrophobicity of a ten to fifteen residue core sequence, which surprisingly does not show any sequence conservation. the functionality of the signals is only marginally impaired by amino-acid exchanges, as long as the hydrophobic core is not affected. exchanges within the core can in contrast totally impede signal recognition, even when leading to a higher hydrophobicity. a closer look at the nature of the six moss signal peptides analysed in this study revealed a high percentage of the hydrophobic amino acids l, f, i, m, v and w in the central part of the signal peptides, even though the hydrophobic stretches vary in length and position. n-terminal of the hydrophobic centre positively charged residues have been proposed. except for ppcalp, all of the moss signal sequence n-termini contain at least one arginine residue. for the signal peptide of ppcalp, which has no positively charged residue, the initiation methionine could perhaps compensate the lack of arginine. all of the investigated signal sequences follow the rule, which means that the two amino acids at position one and three previous to the cleavage site have to be small and neutral. the hmm cleavage site prediction from signalp showed a rather low probability for the signal sequences of pppme and of ppcalp. additionally, in case of ppcalp, the signalp-neural network predicted a signal peptide length of amino acids instead of amino acids which was predicted with signalp-hmm and used in our study. however, experimental data showed that the sequences for both ppcalp and pppme were not only functional, but resulted in a very efficient secretion of the rhvegf protein. in addition, cleavage site prediction of asp and vsp was verified experimentally. the analysis revealed that secretion dropped drastically when the last amino acid of a correctly predicted signal was deleted. the average length of eukaryotic signal peptides was calculated to be amino acids, whereas the average length of analysed physcomitrella signal peptides is residues. although some correlations between the analysed physcomitrella signal peptides and other eukaryotic secretion signals can be found, the variations within the amino acid composition remain high. a functional role of additional, most likely structural determinants of signal peptides is assumed. previous studies evaluating endogenous vs. heterologous signal sequences in several species of the plant and animal kingdoms revealed for the majority a higher efficiency of kingdom-specific signals. as secretion is one of the bottlenecks when establishing a production system, we aimed to define highly efficient signals to transport the recombinant proteins to the culture medium of the moss bioreactor system. thus we decided to compare six endogenous moss signal peptides plus two heterologous sequences from human origin. all of the moss signals turned out to drive secretion more efficiently than the human signal peptides. two signals, the moss-derived asp and the human vsp, were analysed in more detail with two different reporters, rgst and rhvegf, respectively. by secretion of the recombinant reporter protein gst we could show a threefold increase in extracellular accumulation with the asp sequence compared to the human vsp. with the moss secretion signal, extracellular rhvegf amount was up to fivefold more. surprisingly, we did not find an increased intracellular accumulation in case of the human signal peptide. amongst several possible alternatives, our preferred explanation is that recognition by the srp and maybe er-insertion of the human signal peptide is suboptimal thus decreasing the translation rate of the recombinant protein. the differences in secretion efficiency clearly prove that besides hydrophobicity, some additional features, that may have a species-specific character, play a crucial role in signal recognition. as it is known that srp occupies the ef-binding-site of the ribosome and thereby temporarily arrests translation, the cause for a reduced production rate with the human signal could be found in a hindered dissociation of srp after docking of the translation complex to the translocon. the second important interaction of the nascent signal peptide is performed with er-membrane lipids as well as the translocon itself. this interaction could demand unknown structural features of a signal peptide, which are not completely cross-host compatible, as mammalian and plant membranes differ significantly in their lipid composition. however, besides a translational inhibition caused by hindered srp-dissociation, alternative explanations for the worse performance of the human signals do exist: the protein in part may fail to be properly folded and is subsequently degraded. alternatively, transcription of that particular construct may be inefficient. the only possible determinant for such events can be found in the relatively short signal peptide sequences themselves, as all used vectors do not differ in any other detail. apart from the above-mentioned translational inhibition, additional influence of the signal peptide on recombinant protein stability could only take place during, or shortly after er-insertion. when the translation of the protein is completed, the signal peptides are cleaved. thereafter, recombinant products do not differ in any residue and can therefore not exhibit different stabilities. regulatory, post-targeting functions of cleaved signals are proposed by other authors and can therefore not be completely excluded in our case.  even if the structural background remains to be elucidated, our results demonstrate that the six moss sequences are more efficient than the two human ones used in our study in promoting secretion of recombinant human vegf. in the interplay with optimised gene expression and product stabilisation, the utilisation of moss-endogenous secretion signals could greatly enhance the productivity of the moss-bioreactor system. 
efficient targeting to appropriate cell organelles is one of the bottlenecks for the production of recombinant proteins in plant systems. we therefore used fusions of the different signals to heterologous reporter sequences for transient transfection of moss cells and measured the extra- and intracellular accumulation of the recombinant proteins rhvegf and gst, respectively. from the distribution of extra- and intracellular recombinant proteins, we suggest translational inhibition during the signal recognition particle-cycle as the most probable of several possible explanations for the decreased extracellular accumulation with the human signals. though the molecular details of this effect remain to be elucidated, our results will contribute to the improvement of molecular farming systems. in this work, we report on the supremacy of moss secretion signals over the utilised heterologous ones within the moss-bioreactor system. a common practice is to use the native secretory signal peptide of the heterologous protein to be produced. though general features of secretion signals are conserved between plants and animals, the broad sequence variability among signal peptides suggests differing efficiency of signal peptide recognition. aiming to improve secretion in moss bioreactors, we quantitatively compared the efficiency of two human signal peptides and six signals from recently isolated moss proteins. our data demonstrates an up to fivefold higher secretion efficiency with endogenous moss signals compared to the two utilised human signal peptides.
 efficient targeting to appropriate cell organelles is one of the bottlenecks for the production of recombinant proteins in plant systems. a common practice is to use the native secretory signal peptide of the heterologous protein to be produced. though general features of secretion signals are conserved between plants and animals, the broad sequence variability among signal peptides suggests differing efficiency of signal peptide recognition.  aiming to improve secretion in moss bioreactors, we quantitatively compared the efficiency of two human signal peptides and six signals from recently isolated moss proteins. we therefore used fusions of the different signals to heterologous reporter sequences for transient transfection of moss cells and measured the extra- and intracellular accumulation of the recombinant proteins rhvegf and gst, respectively. our data demonstrates an up to fivefold higher secretion efficiency with endogenous moss signals compared to the two utilised human signal peptides.  from the distribution of extra- and intracellular recombinant proteins, we suggest translational inhibition during the signal recognition particle-cycle as the most probable of several possible explanations for the decreased extracellular accumulation with the human signals. in this work, we report on the supremacy of moss secretion signals over the utilised heterologous ones within the moss-bioreactor system. though the molecular details of this effect remain to be elucidated, our results will contribute to the improvement of molecular farming systems. 