BACKGROUND
polyhydroxyalkanoate  granules  are formed inside bacterial cells based on the activity and biochemical properties of the pha synthases and specific biosynthesis enzymes which are involved in pha precursor supply  <cit> .
biologically, pha serves as a reserve material.
the pha granule core is composed of pha and the surface of a phospholipid membrane with embedded or attached proteins.
amphipathic phasin proteins are one group of proteins specifically and hydrophobically interacting with the pha core .
the functional role of the phasins impacting on pha granule structure has been studied in detail  <cit> .
phasins and their fusion proteins have been increasingly considered for protein production at the pha granule surface  <cit> .
recently, pha synthase engineering enabled production of the beta-galactosidase and gfp fusion proteins, respectively, at the pha granule surface  <cit> .
the gfp-pha synthase fusion even enabled monitoring of in vivo pha granule formation indicating that pha granule formation starts at the cell poles.
only recently, pha granules have been considered as spherical biopolyester particles which can be stably maintained outside the bacterial cell exerting a size range from about  <dig> nm to several μm  <cit> .
here we co-expressed the pha biosynthesis operon from cupriavidus necator with a hybrid gene encoding a phasin fusion protein in escherichia coli in order to mediate the formation of pha granules efficiently displaying the respective fusion partner.
in this study, eukaryotic antigen displaying pha granules were designed and their application performance with respect to diagnostic applications using fluorescence activated cell sorting  was evaluated.
the displayed myelin oligodendrocyte glycoprotein  was used as an example depicting diagnostic analysis of the autoimmune disease multiple sclerosis .
it was for the first time observed that surface-engineered and antigen displaying pha granules can be efficiently used for facs based diagnostics.
thus designed pha granules, which combine cheap one step production with facilitated folding of proteins, might in future replace commercial beads.
RESULTS
production of phap fused to il <dig> or mog at the pha granule surface
the two immunologically and medically relevant proteins mog and il <dig> derived from mus musculus were each produced as phap fusion protein via construction of the respective hybrid gene.
the dna sequences encoding either mog or il <dig> were optimized with respect to the codon usage of e. coli and purchased as synthetic dna fragment .
an enterokinase recognition site plus six histidine residues were inserted as linker region between phap and il <dig> or mog in order to facilitate independent folding of the fusion partner and to enable specific removal of the antigen from the fusion partner.
plasmids pbhr68-phap-mog and pbhr68-phap-il <dig>  which both comprise the entire phb biosynthesis operon in addition to the respective hybrid gene encoding the phap fusion protein, were introduced into e. coli xl <dig> blue .
the respective recombinant strains were cultivated and pha granules isolated.
proteins attached to these granules were separated by sds-page and prominent proteins were subjected to peptide fingerprinting using maldi-tof/ms.
plasmid pbhr68-phap-mog mediated the production of pha granules showing four prominent proteins with apparent molecular weights of  <dig> kda,  <dig> kda,  <dig> kda and  <dig> kda .
the  <dig> kda and the  <dig> kda protein were strongly overproduced and identified as phap-mog fusion proteins using maldi-tof/ms analysis .
both proteins were subjected to n terminal amino acid sequencing and the  <dig> kda phap-mog fusion showed the expected n terminal amino acid sequence miltp, whereas the more abundant  <dig> kda phap-mog showed an n terminus of mtmitp.
the additional two more prominent proteins were subjected to maldi-tof/ms analysis and identified the  <dig> kda protein as ef-tu , but no assignment of the  <dig> kda could be obtained .
plasmid pbhr68-phap-il <dig> mediated the production of pha granules showing two prominent and strongly overproduced proteins with apparent molecular weights of  <dig> kda and  <dig> kda.
both proteins were identified as phap-il <dig> fusion proteins using maldi-tof/ms analysis  and were subjected to n terminal sequencing and the  <dig> kda phap-mog fusion showed the expected n terminus of miltp, whereas the more abundant  <dig> kda phap-mog showed an n terminus of mtmitp.
batch cultivations led to the production of about  <dig> ×  <dig> pha granules/l cultivation broth corresponding to about  <dig>  g protein displayed at the pha granules surface.
native il <dig> and mog proteins were displayed on pha granules
to test whether il <dig> and mog were expressed in native form on the surface of the respective pha granules, antibodies that recognize correctly folded il <dig>  or mog  and facs technology were used  <cit> .
the size of pha granules produced in e. coli has been reported to be about 100– <dig> nm  <cit> , and were detected in the forward scatter  and 90° side scatter  detectors set to list mode at a 256-channel resolution .
pha granules were incubated with unlabelled mouse anti-mog antibodies, followed by apc-labelled anti-mouse igg antibodies, or with pe-labelled anti-il <dig> antibodies.
the fluorescent intensity was then measured using facs technology.
results show that the monoclonal antibodies specifically recognized pha granules displaying the specific antigen, but not an un-related antigen .
both pc <dig> and 8-18c <dig> recognize only the respective native protein indicating that correctly folded il- <dig> or mog proteins were formed at the surface of the respective pha granules.
these pha granules showed consistent performance in facs analysis at least one year when stored at 4°c.
phap-il <dig> and phap-mog fusion proteins containing the asp-asp-asp-asp-lys recognition sequence are cleavable with enterokinase
to enable removal and/or purification of recombinant eukaryotic proteins, which form inactive inclusion bodies in e. coli, an enterokinase recognition sequence encoding for the asp-asp-asp-asp-lys peptide was incorporated immediately downstream of phap.
to determine if the recognition sequence was readily accessible, phap-il <dig> and phap-mog containing pha granules were incubated for  <dig> h with enterokinase.
samples of phap-il <dig> and phap-mog granules were removed before, after  <dig> h and after  <dig> h of incubation with enterokinase to determine the level of native protein remaining at the surface of the respective pha granule.
phap-il <dig> and phap-mog granules were incubated with pe-conjugated anti-il <dig> and samples analysed by facs .
a similar pattern of decreased protein levels after incubation with enterokinase was found with phap-mog granules incubated with anti-mog monoclonal antibodies .
the level of il <dig> was reduced by ~80% after  <dig> h incubation and after  <dig> h incubation no significant levels of il <dig> protein were detectable at the surface of the granules .
overall, results shown in figures  <dig> and  <dig> demonstrate that correctly folded recombinant proteins can be expressed as a fusion protein together with phap and that the fusion partner can be completely removed by enterokinase treatment.
pha granules displaying phap-mog fusion proteins were used in facs-based assays to detect antigen-specific serum antibodies
since pha granules can be detected using facs technology, we tested whether phap-mog granules could be used to detect and quantify the level of antigen-specific antibodies from immunized animals.
for this purpose, mice were immunized with recombinant mog or ova protein in cfa to induce an antibody response to mog and ova, respectively.
twenty-eight days post immunization, mice were tail-bled and immune sera collected.
sera from five different mice were pooled and tested for binding to phap-mog granules.
results in figure 5a showed that mog-immunized, but not ova-immunized, anti-sera contained antigen-specific igg antibodies that recognized the phap-mog fusion protein.
the sera could be diluted >1: <dig> fold and still showed a significant binding of igg to the phap-mog granules .
sera from ova-immunized mice did not bind significantly to phap-mog granules.
however, ova immunized mice produced ova-specific igg, as micro-titre wells coated with ova, but not with mog, showed bound igg which was easily detectable using elisa .
these results illustrate that recombinant eukaryotic proteins displayed with phap as a fusion protein can be applied using facs technology to detect and quantify the level of antigen-specific antibodies from vaccinated animals.
discussion
in this study, the surface of pha granules produced by recombinant e. coli was subjected to protein engineering to display natively folded eukaryotic proteins, which serve as antigens in diagnostics .
both chosen eukaryotic proteins mog and the cytokine il- <dig> form inclusion bodies upon production in e. coli  <cit> .
a microbial protein production system avoiding the formation of inclusion bodies would have a huge advantage by circumventing the tedious process of solubilization and refolding.
the pha granule attached structural protein phap was n terminally fused to either mog or il <dig> .
a strong overproduction under lac promoter control of the respective fusion proteins could be observed .
the fusion proteins could be identified by peptide fingerprinting using maldi-tof/ms analysis and by n terminal amino acid sequencing.
interestingly, the fusion proteins appeared in two versions with different apparent molecular weight.
the smaller molecular weight version of the fusion protein showed the theoretical molecular weight of the respective fusion protein.
the n terminal sequence  of the larger molecular weight version suggested that the n terminal  <dig> amino acid residues  of lacz were fused to the n terminus of phap fusion protein based on start codon upstream of the start codon of the hybrid gene encoding the phap-mog or phap-il <dig>  this was also in accordance with the apparent molecular weight difference of  <dig>  kda between the two versions.
interestingly, the lacz'-phap-mog/il <dig> fusion proteins were more abundant than the respective single fusions .
these data suggest that n terminal fusions to phap do not interfere with the binding to the pha core of pha granules and that the copy numbers can be even enhanced.
this is unexpected considering the current phasin protein model  <cit> .
the presence of the two e. coli proteins  <dig> kda and ef-tu at the mog displaying pha granule surface might be due to an unspecific interaction with pha granule surface, which has been already observed elsewhere  <cit> .
in contrast to a previous study, where multiple copies of phap were required for binding of prokaryotic protein fusions, a single copy was sufficient using an enterokinase site linker and eukaryotic proteins  <cit> .
engineered pha granules were subjected to facs analysis using antibodies specifically recognizing the respective native protein folds, which showed the display of the natively folded eukaryotic proteins mog and il <dig> at the pha granule surface as well as the applicability of designed pha granules for facs-based diagnostics .
although mog and il <dig> are secreted proteins, the derived protein domains were properly folded attached to the pha granule in the reducing cytosol of e. coli while avoiding the formation of protein inclusion bodies.
although e. coli strains  are available, which provide an oxidative cytosol, protein overproduction often leads to inclusion body formation.
enterokinase treatment of mog or il <dig> displaying pha granules and the complete release of the antigen suggested that the respective antigen was exposed at the pha granule surface .
this surface exposure enables efficient antigen specific antibody binding.
to evaluate the suitability of mog or il <dig> displaying pha granules for qualitative and quantitative facs-based antibody detection, mice were immunized with mog or ova  and induction of specific antibody production was assessed and confirmed by elisa .
these mog or ova antisera were analysed using the mog displaying pha granules and facs technology, which clearly indicated that anti-mog antibodies can be specifically detected at least up to an antisera dilution of 1: <dig>  .
in this study, it was demonstrated that eukaryotic proteins can be functionally displayed at the pha granule surface using protein engineering of phap.
evidence was provided that the fusion proteins  were exposed at the pha granule and thus pha granules could be used to capture antibodies.
this feature in combination with the particle properties of the pha granules led to an outstanding performance in facs based diagnostics, particularly considering the signal to noise ratio and dynamic range of antibody detection.
it was recently shown that zz domain displaying pha granules, which were produced via protein engineering of the pha synthase, were suitable for igg purification  <cit> .
when compared to commercial beads, engineered pha granules showed increased sensitivity with similar distribution of signal intensities in facs.
overall, protein engineering of pha granule surface proteins provides a novel molecular tool for the display of antigens for facs-based diagnostics.
CONCLUSIONS
in this study, it was demonstrated that correctly folded eukaryotic proteins can be abundantly produced at the pha granule surface as phasin fusion proteins.
isolated pha granules displaying the respective eukaryotic proteins could be used as beads for specific and sensitive antibody detection using facs technology.
these native antigen displaying pha granules were manufactured by recombinant e. coli without the need of antigen purification and chemical cross-linking to independently produced beads.
often purification of these proteins requires tedious refolding at low efficiency.
the production of functional eukaryotic proteins at the pha granule surface represents a novel in vivo matrix-assisted protein folding system avoiding aggregation of protein folding intermediates.
moreover, pha granules could be stored for at least one year at 4°c without loss of performance in facs supporting their potential use in diagnostic applications.
this work opens up an alternative route for the production of protein displaying beads harnessing nature's capacity to produce spherical polymer beads which surface can be functionalized by engineering of specifically bead associating proteins .
multiple functionality might be easy achievable by co-expression of various hybrid genes suggesting that this biotechnological bead production strategy might represent a versatile platform technology.

 polyhydroxyalkanoate granules are formed inside bacterial cells based on the activity and biochemical properties of the pha synthases and specific biosynthesis enzymes which are involved in pha precursor supply. biologically, pha serves as a reserve material. the pha granule core is composed of pha and the surface of a phospholipid membrane with embedded or attached proteins. amphipathic phasin proteins are one group of proteins specifically and hydrophobically interacting with the pha core. the functional role of the phasins impacting on pha granule structure has been studied in detail. phasins and their fusion proteins have been increasingly considered for protein production at the pha granule surface. recently, pha synthase engineering enabled production of the beta-galactosidase and gfp fusion proteins, respectively, at the pha granule surface. the gfp-pha synthase fusion even enabled monitoring of in vivo pha granule formation indicating that pha granule formation starts at the cell poles. only recently, pha granules have been considered as spherical biopolyester particles which can be stably maintained outside the bacterial cell exerting a size range from about nm to several μm. here we co-expressed the pha biosynthesis operon from cupriavidus necator with a hybrid gene encoding a phasin fusion protein in escherichia coli in order to mediate the formation of pha granules efficiently displaying the respective fusion partner. in this study, eukaryotic antigen displaying pha granules were designed and their application performance with respect to diagnostic applications using fluorescence activated cell sorting was evaluated. the displayed myelin oligodendrocyte glycoprotein was used as an example depicting diagnostic analysis of the autoimmune disease multiple sclerosis. it was for the first time observed that surface-engineered and antigen displaying pha granules can be efficiently used for facs based diagnostics. thus designed pha granules, which combine cheap one step production with facilitated folding of proteins, might in future replace commercial beads.  production of phap fused to il or mog at the pha granule surface the two immunologically and medically relevant proteins mog and il derived from mus musculus were each produced as phap fusion protein via construction of the respective hybrid gene. the dna sequences encoding either mog or il were optimized with respect to the codon usage of e. coli and purchased as synthetic dna fragment. an enterokinase recognition site plus six histidine residues were inserted as linker region between phap and il or mog in order to facilitate independent folding of the fusion partner and to enable specific removal of the antigen from the fusion partner. plasmids pbhr68-phap-mog and pbhr68-phap-il which both comprise the entire phb biosynthesis operon in addition to the respective hybrid gene encoding the phap fusion protein, were introduced into e. coli xl blue. the respective recombinant strains were cultivated and pha granules isolated. proteins attached to these granules were separated by sds-page and prominent proteins were subjected to peptide fingerprinting using maldi-tof/ms. plasmid pbhr68-phap-mog mediated the production of pha granules showing four prominent proteins with apparent molecular weights of kda, kda, kda and kda. the kda and the kda protein were strongly overproduced and identified as phap-mog fusion proteins using maldi-tof/ms analysis. both proteins were subjected to n terminal amino acid sequencing and the kda phap-mog fusion showed the expected n terminal amino acid sequence miltp, whereas the more abundant kda phap-mog showed an n terminus of mtmitp. the additional two more prominent proteins were subjected to maldi-tof/ms analysis and identified the kda protein as ef-tu, but no assignment of the kda could be obtained. plasmid pbhr68-phap-il mediated the production of pha granules showing two prominent and strongly overproduced proteins with apparent molecular weights of kda and kda. both proteins were identified as phap-il fusion proteins using maldi-tof/ms analysis and were subjected to n terminal sequencing and the kda phap-mog fusion showed the expected n terminus of miltp, whereas the more abundant kda phap-mog showed an n terminus of mtmitp. batch cultivations led to the production of about × pha granules/l cultivation broth corresponding to about g protein displayed at the pha granules surface. native il and mog proteins were displayed on pha granules to test whether il and mog were expressed in native form on the surface of the respective pha granules, antibodies that recognize correctly folded il or mog and facs technology were used. the size of pha granules produced in e. coli has been reported to be about 100– nm, and were detected in the forward scatter and 90° side scatter detectors set to list mode at a 256-channel resolution. pha granules were incubated with unlabelled mouse anti-mog antibodies, followed by apc-labelled anti-mouse igg antibodies, or with pe-labelled anti-il antibodies. the fluorescent intensity was then measured using facs technology. results show that the monoclonal antibodies specifically recognized pha granules displaying the specific antigen, but not an un-related antigen. both pc and 8-18c recognize only the respective native protein indicating that correctly folded il- or mog proteins were formed at the surface of the respective pha granules. these pha granules showed consistent performance in facs analysis at least one year when stored at 4°c. phap-il and phap-mog fusion proteins containing the asp-asp-asp-asp-lys recognition sequence are cleavable with enterokinase to enable removal and/or purification of recombinant eukaryotic proteins, which form inactive inclusion bodies in e. coli, an enterokinase recognition sequence encoding for the asp-asp-asp-asp-lys peptide was incorporated immediately downstream of phap. to determine if the recognition sequence was readily accessible, phap-il and phap-mog containing pha granules were incubated for h with enterokinase. samples of phap-il and phap-mog granules were removed before, after h and after h of incubation with enterokinase to determine the level of native protein remaining at the surface of the respective pha granule. phap-il and phap-mog granules were incubated with pe-conjugated anti-il and samples analysed by facs. a similar pattern of decreased protein levels after incubation with enterokinase was found with phap-mog granules incubated with anti-mog monoclonal antibodies. the level of il was reduced by ~80% after h incubation and after h incubation no significant levels of il protein were detectable at the surface of the granules. overall, results shown in figures and demonstrate that correctly folded recombinant proteins can be expressed as a fusion protein together with phap and that the fusion partner can be completely removed by enterokinase treatment. pha granules displaying phap-mog fusion proteins were used in facs-based assays to detect antigen-specific serum antibodies since pha granules can be detected using facs technology, we tested whether phap-mog granules could be used to detect and quantify the level of antigen-specific antibodies from immunized animals. for this purpose, mice were immunized with recombinant mog or ova protein in cfa to induce an antibody response to mog and ova, respectively. twenty-eight days post immunization, mice were tail-bled and immune sera collected. sera from five different mice were pooled and tested for binding to phap-mog granules. results in figure 5a showed that mog-immunized, but not ova-immunized, anti-sera contained antigen-specific igg antibodies that recognized the phap-mog fusion protein. the sera could be diluted >1: fold and still showed a significant binding of igg to the phap-mog granules. sera from ova-immunized mice did not bind significantly to phap-mog granules. however, ova immunized mice produced ova-specific igg, as micro-titre wells coated with ova, but not with mog, showed bound igg which was easily detectable using elisa. these results illustrate that recombinant eukaryotic proteins displayed with phap as a fusion protein can be applied using facs technology to detect and quantify the level of antigen-specific antibodies from vaccinated animals. discussion in this study, the surface of pha granules produced by recombinant e. coli was subjected to protein engineering to display natively folded eukaryotic proteins, which serve as antigens in diagnostics. both chosen eukaryotic proteins mog and the cytokine il- form inclusion bodies upon production in e. coli. a microbial protein production system avoiding the formation of inclusion bodies would have a huge advantage by circumventing the tedious process of solubilization and refolding. the pha granule attached structural protein phap was n terminally fused to either mog or il. a strong overproduction under lac promoter control of the respective fusion proteins could be observed. the fusion proteins could be identified by peptide fingerprinting using maldi-tof/ms analysis and by n terminal amino acid sequencing. interestingly, the fusion proteins appeared in two versions with different apparent molecular weight. the smaller molecular weight version of the fusion protein showed the theoretical molecular weight of the respective fusion protein. the n terminal sequence of the larger molecular weight version suggested that the n terminal amino acid residues of lacz were fused to the n terminus of phap fusion protein based on start codon upstream of the start codon of the hybrid gene encoding the phap-mog or phap-il this was also in accordance with the apparent molecular weight difference of kda between the two versions. interestingly, the lacz'-phap-mog/il fusion proteins were more abundant than the respective single fusions. these data suggest that n terminal fusions to phap do not interfere with the binding to the pha core of pha granules and that the copy numbers can be even enhanced. this is unexpected considering the current phasin protein model. the presence of the two e. coli proteins kda and ef-tu at the mog displaying pha granule surface might be due to an unspecific interaction with pha granule surface, which has been already observed elsewhere. in contrast to a previous study, where multiple copies of phap were required for binding of prokaryotic protein fusions, a single copy was sufficient using an enterokinase site linker and eukaryotic proteins. engineered pha granules were subjected to facs analysis using antibodies specifically recognizing the respective native protein folds, which showed the display of the natively folded eukaryotic proteins mog and il at the pha granule surface as well as the applicability of designed pha granules for facs-based diagnostics. although mog and il are secreted proteins, the derived protein domains were properly folded attached to the pha granule in the reducing cytosol of e. coli while avoiding the formation of protein inclusion bodies. although e. coli strains are available, which provide an oxidative cytosol, protein overproduction often leads to inclusion body formation. enterokinase treatment of mog or il displaying pha granules and the complete release of the antigen suggested that the respective antigen was exposed at the pha granule surface. this surface exposure enables efficient antigen specific antibody binding. to evaluate the suitability of mog or il displaying pha granules for qualitative and quantitative facs-based antibody detection, mice were immunized with mog or ova and induction of specific antibody production was assessed and confirmed by elisa. these mog or ova antisera were analysed using the mog displaying pha granules and facs technology, which clearly indicated that anti-mog antibodies can be specifically detected at least up to an antisera dilution of 1:. in this study, it was demonstrated that eukaryotic proteins can be functionally displayed at the pha granule surface using protein engineering of phap. evidence was provided that the fusion proteins were exposed at the pha granule and thus pha granules could be used to capture antibodies. this feature in combination with the particle properties of the pha granules led to an outstanding performance in facs based diagnostics, particularly considering the signal to noise ratio and dynamic range of antibody detection. it was recently shown that zz domain displaying pha granules, which were produced via protein engineering of the pha synthase, were suitable for igg purification. when compared to commercial beads, engineered pha granules showed increased sensitivity with similar distribution of signal intensities in facs. overall, protein engineering of pha granule surface proteins provides a novel molecular tool for the display of antigens for facs-based diagnostics.  in this study, it was demonstrated that correctly folded eukaryotic proteins can be abundantly produced at the pha granule surface as phasin fusion proteins. isolated pha granules displaying the respective eukaryotic proteins could be used as beads for specific and sensitive antibody detection using facs technology. these native antigen displaying pha granules were manufactured by recombinant e. coli without the need of antigen purification and chemical cross-linking to independently produced beads. often purification of these proteins requires tedious refolding at low efficiency. the production of functional eukaryotic proteins at the pha granule surface represents a novel in vivo matrix-assisted protein folding system avoiding aggregation of protein folding intermediates. moreover, pha granules could be stored for at least one year at 4°c without loss of performance in facs supporting their potential use in diagnostic applications. this work opens up an alternative route for the production of protein displaying beads harnessing nature's capacity to produce spherical polymer beads which surface can be functionalized by engineering of specifically bead associating proteins. multiple functionality might be easy achievable by co-expression of various hybrid genes suggesting that this biotechnological bead production strategy might represent a versatile platform technology. 
beads displaying a specific antigen are used to bind antibodies which are then fluorescently labelled using secondary antibodies. hybrid genes were constructed, which encode either the mouse interleukin- or the myelin oligodendrocyte glycoprotein fused via an enterokinase site providing linker region to the c terminus of the pha granule associated protein phap, respectively. mog and il fusion proteins were abundantly attached to pha granules and were identified by maldi-tof/ms analysis and n terminal sequencing. a more abundant second fusion protein of either mog or il resulted from an additional n terminal fusion, which did surprisingly not interfere with attachment to pha granule. pha granules displaying either il or mog were used for facs using monoclonal anti-il or anti-mog antibodies conjugated to a fluorescent dye. enterokinase treatment of il displaying pha granules enabled removal of il as monitored by facs analysis. mice were immunized with either mog or ova and the respective sera were analysed using mog-displaying pha granules and facs analysis showing a specific and sensitive detection of antigen-specific antibodies within a wide dynamic range. e. coli can be genetically engineered to produce pha granules displaying correctly folded eukaryotic proteins and which can be applied as beads in facs based diagnostics. since pha granule formation and protein attachment occurs in one step already inside the bacterial cell, microbial production could be a cheap and efficient alternative to commercial beads. the hybrid genes were expressed in pha-accumulating recombinant e. coli. as an alternative, we genetically engineered escherichia coli to produce biopolyester granules displaying diagnostically relevant antigens in their native conformation and suitable for facs analysis. facs analysis showed significant and specific binding of respective antibodies.
 fluorescence activated cell sorting is a powerful technique for the qualitative and quantitative detection of biomolecules used widely in both basic research and clinical diagnostic applications. beads displaying a specific antigen are used to bind antibodies which are then fluorescently labelled using secondary antibodies. as the individual suspension bead passes through the sensing region of the facs machine, fluorescent signals are acquired and analysed. currently, antigens are tediously purified and chemically cross-linked to preformed beads. purification and coupling of proteins often renders them inactive and they will not be displayed in its native configuration. as an alternative, we genetically engineered escherichia coli to produce biopolyester granules displaying diagnostically relevant antigens in their native conformation and suitable for facs analysis.  hybrid genes were constructed, which encode either the mouse interleukin- or the myelin oligodendrocyte glycoprotein fused via an enterokinase site providing linker region to the c terminus of the pha granule associated protein phap, respectively. the hybrid genes were expressed in pha-accumulating recombinant e. coli. mog and il fusion proteins were abundantly attached to pha granules and were identified by maldi-tof/ms analysis and n terminal sequencing. a more abundant second fusion protein of either mog or il resulted from an additional n terminal fusion, which did surprisingly not interfere with attachment to pha granule. pha granules displaying either il or mog were used for facs using monoclonal anti-il or anti-mog antibodies conjugated to a fluorescent dye. facs analysis showed significant and specific binding of respective antibodies. enterokinase treatment of il displaying pha granules enabled removal of il as monitored by facs analysis. mice were immunized with either mog or ova and the respective sera were analysed using mog-displaying pha granules and facs analysis showing a specific and sensitive detection of antigen-specific antibodies within a wide dynamic range.  e. coli can be genetically engineered to produce pha granules displaying correctly folded eukaryotic proteins and which can be applied as beads in facs based diagnostics. since pha granule formation and protein attachment occurs in one step already inside the bacterial cell, microbial production could be a cheap and efficient alternative to commercial beads. 