BACKGROUND
the bacteriophage m <dig> is assembled during a secretion process in the cytoplasmic membrane of escherichia coli.
membrane inserted phage proteins contact the single stranded phage dna in an helical array and pass through the outer membrane by a porin-like structure composed of gp <dig>  <cit> .
in the inner membrane a protein complex probably consisting of gp <dig>  gp <dig> and thioredoxin catalyses the assembly process  <cit> .
first, membrane inserted gp <dig> and gp <dig> proteins form a tip structure  <cit>  that is extended by a multiple array of gp <dig> proteins, the major coat protein.
gp <dig> is synthesised as a precursor protein, termed procoat, that is inserted into the inner membrane by the yidc protein  <cit>  and is then processed by leader peptidase  <cit> .
after processing, the transmembrane coat proteins assemble into oligomers and bind to the viral dna forming the nascent phage filament  <cit> .
this filament traverses the outer membrane through the gp <dig> complex  <cit> .
finally, the membrane inserted gp <dig> and gp <dig> proteins are assembled onto the extruding phage at the proximal end of the virion terminating phage assembly.
the gp <dig> protein has been extensively used for the phage display technology.
since gp <dig> is engaged in the adsorption of the phage onto the host cell certain restrictions on the infectivity of the modified phage have to be encountered  <cit> .
this might be different for gp <dig> modifications since this protein is localized at the distal end of the filamentous phage particle.
previously, it has been shown that gp <dig> is accessible in the phage particle  <cit> .
therefore, gp <dig> might be a good target for phage display technology  <cit> .
in addition, an attractive idea is to have both ends supplied with functional peptide moieties applicable as molecular measures or bifunctional binders.
gp <dig> is a  <dig> amino acid long protein that is synthesised without a signal sequence.
it is thought that the membrane-inserted protein displays its n-terminus into the periplasm.
however, the first amino-terminal  <dig> residues are hydrophobic and it is questionable whether the protein spans the entire bilayer.
one possibility to explore this is to fuse hydrophilic peptides onto the n-terminus.
when these modified gp <dig> proteins are inserted into the membrane their amino-terminal region can be analysed whether they are exposed in the periplasm.
therefore, we have fused short antigenic peptides to the n-terminus of gp <dig> between the residues  <dig> and  <dig>  they extend the protein by  <dig> to  <dig> amino acid residues.
the proteins are inserted into the membrane and efficiently assemble onto phage progeny particles since they can substitute for the wild-type protein.
also, the antigenic epitopes are detectable with gold-labelled antibodies by electron microscopy.
RESULTS
antigenic epitopes at the n-terminus of m <dig> gp9
to study the assembly of m <dig> gp <dig>  genetic variants were constructed that extend the n-terminal region of the protein with antigenic epitopes.
two antigenic peptides, a  <dig> amino acid residues long t7-tag and a  <dig> residues long ha-tag were fused to the n-terminus of gp <dig>  respectively .
this was performed on a gene  <dig> copy on a pms <dig> plasmid using a unique muni restriction site that was engineered between the codons  <dig> and  <dig> to generate gp9muni.
into this site, dna fragments encoding the tag sequences were introduced.
in addition, longer fragments were introduced which encode two copies of the antigenic tag sequences, resulting in additional  <dig> and  <dig> residues in pms-g9-dt <dig> and pms-g9-dha, respectively.
then, the functionality of the modified proteins was tested by complementation of an m13am <dig> phage infection .
e. coli k <dig> bearing the corresponding plasmid was grown overnight in lb medium and plated with top agar containing  <dig> mm iptg.
after solidification of the top agar  <dig> μl of a phage suspension was applied on top of the agar.
plaque formation was observed after incubation at 37°c overnight.
when the cells with pms-g9-ha were infected with m13am <dig> clear plaques with a turbid zone were visible on the bacterial lawn .
whereas no plaques appeared with the k <dig> cells containing the pms plasmid , pms-g7/ <dig> transformed cells showed plaque formation down to the 105-fold dilution step .
in the absence of iptg  plaque formation was observed at the 104-fold dilution which is most likely due to a low expression or to recombination events.
when k <dig> cells with the pms-g9-t <dig>  or with pms-g9-ha  were used plaque formation was evident down to the 105-fold dilution step.
similarly, the plasmids encoding the double tags  showed efficient plaque formation, as it was observed on the plates with the suppressor containing e. coli k <dig> cells .
these results suggest that the gp <dig> variants expressing the epitope-tagged proteins are functional and allow normal phage propagation.
expression of the modified gp <dig> protein in e. coli
the plasmid-encoded gp <dig> variants were analysed for expression in e. coli k <dig>  the cells were grown at 37°c to the early exponential phase in m <dig> minimal medium.
protein expression was induced by adding  <dig> mm iptg and after  <dig> min the newly synthesised proteins were pulse-labelled for  <dig> min with 35s-methionine.
the total bacterial proteins were tca precipitated to remove the non-incorporated 35s-methionine and immunoprecipitated using an antiserum to the t <dig> tag or to the ha tag, respectively .
since gp <dig> is a very small protein of  <dig> amino acids containing only two methionines the protein band on a sds tricine page is difficult to visualise.
when comparing the protein pattern of cells expressing gp9-t <dig>  with cells containing the empty plasmid  a protein band of about  <dig>  kda was observed.
also a weak band of gp9-ha  was visible on the gel.
the size of the protein was estimated in relation of the major coat protein gp <dig> shown in lane  <dig>  since the  <dig> amino acid residues long gp <dig> has a molecular weight of  <dig>  kda, the gp9-t <dig> with  <dig> residues and gp9-ha with  <dig> residues are proteins of very similar molecular weight.
membrane insertion of gp9-t7
the membrane insertion of gp <dig> with the n-terminal t <dig> tag was analysed in e. coli k <dig> cells bearing the pms-g9-t <dig> plasmid.
the gp9-t <dig> protein was expressed as described above.
the cells were converted to spheroplasts and analysed by protease mapping .
the protein immunoprecipitated with antiserum to the t <dig> tag was readily digested by proteinase k added to the outside of the spheroplasts .
this suggests that the antigenic tag of gp <dig> was accessible to the protease at the periplasmic surface, whereas the cytoplasmic groel protein was protected from digestion .
further, the periplasmic portion of the ompa protein was digested by the proteinase k  confirming the proteolytic activity.
the insertion of gp9-t <dig> into the membrane was then investigated in e. coli js <dig>  in these cells, the membrane insertase yidc can be depleted when the cells are grown in the presence of glucose  <cit> .
after  <dig> h growth under glucose conditions the cells were pulse-labelled with 35s-methionine for  <dig> min and converted to spheroplasts.
the protease mapping  shows that the yidc depleted cells did not allow the digestion of the t7-epitope at the n-terminus of gp <dig> .
these results suggest that the membrane insertion of gp9-t <dig> is yidc-dependent.
in both cases, the integrity of the spheroplasts was verified by the protection of groel  and the proteolytic activity was corroborated by the accessibility of the ompa protein .
assembly of gp <dig> variant proteins onto phage
assembly of the plasmid-encoded variants onto phage was first followed by dot-blot analysis of phage particles.
m13am <dig> infections in e. coli k <dig> bearing a plasmid coding for one of the gp <dig> variants were performed and the progeny phage were collected and titrated.
equal amounts of phage was applied on nitrocellulose, incubated with antiserum to m <dig> gp <dig>  to t <dig> tag or to the ha tag, respectively.
the reaction with a secondary peroxidase coupled antibody was analysed by chemoluminescence .
whereas the infecting m13am <dig> phage reacted only to the anti gp <dig> serum , the phage grown in cells with pms-g9-t <dig> clearly reacted with the t <dig> serum .
similarly, phage from cells expressing the double tag gp9-dt <dig> also reacted with the serum to the t <dig> tag.
strong signals were obtained with gp <dig> proteins with the ha epitopes  whereas the uninfected k <dig> cells expressing gp9-t <dig> or gp9-ha showed only a low signal in the corresponding supernatants.
this verifies that the plasmid encoded gp <dig> proteins with the epitope tags were efficiently assembled onto the phage particles.
the exposure of the antigenic epitopes on the phage particles was then tested with immunogold .
first, phage was incubated with the respective serum, then with protein a coupled immunogold particles  and applied to coated copper grids.
after staining with 5% phosphotungstic acid  the phage particles were inspected.
several gold nanoparticles were bound to the tip of individual phages either with the t <dig> tag  the double t <dig> tag , or the double ha tag .
the parental m13am <dig> phage, used as a control showed no binding of the gold nanoparticles to the tip .
in contrast, for both complemented phage particles we found that about 30% of the gold nanoparticles were bound to phage particles and about 20% of the phage had a gold nanoparticle bound at the tip.
taken together, the analysis shows that the modified gp <dig> proteins are well exposed and accessible to antibodies.
discussion
the minor coat protein gp <dig> of the filamentous phage m <dig> is exposed from the phage particle and can be modified with short peptides.
here we have shown that peptides of  <dig>   <dig>   <dig> and  <dig> amino acid residues can be incorporated into the amino-terminal region of the protein without interfering with membrane insertion and assembly of the phage.
the epitopes of these peptides are accessible by antibodies and allow binding of gold nanoparticles that can be visualised by electron microscopy.
this implicates that gp <dig> could be used for the phage display methodology allowing a combination with the well-established display of modified gp <dig>  previous experiments have shown that gp <dig> of the closely related fd phage is localised at the distal end, together with gp <dig>  <cit> .
in that study, it was also shown, that the gp <dig> protein is exposed to the surface in contrast to gp <dig>  however, a fusion with glutathione-s-transferase  did not support phage production suggesting that membrane insertion or phage assembly is inhibited.
since gst is a folded structure of about  <dig> kda we tested smaller fusion proteins that may be tolerated for membrane insertion and phage assembly.
by introducing short antigenic sequences between the amino acid residues  <dig> and  <dig> of gp <dig> on a plasmid membrane insertion and phage assembly was followed.
also, longer fusions consisting of  <dig> and  <dig> additional residues that code for two tandem tags were constructed.
intriguingly, all gp <dig> fusion proteins complement an amber- <dig> phage infection and lead to progeny production up to wild-type levels.
when the phage progeny particles were analysed for the presentation of their antigenic epitopes we observed by dot-blot analysis  and immunogold labelling  a clearly positive response.
we conclude that the amino-terminal end of gp <dig> is capable to accept modifications and provides a new possibility for phage display.
the extended amino-terminal region with an antigenic tag allowed the investigation of the membrane insertion of gp <dig> in detail.
previously, it had been shown by ftir spectroscopy that the membrane-inserted protein has a high α-helical conformation and adopts a transmembrane conformation  <cit> .
in a short pulse, the synthesised gp <dig> was radioactively labelled and analysed for membrane insertion by protease added to the outside of the membrane .
indeed, the protease removed the antigenic tag at the n-terminus of gp <dig>  whereas the cytoplasmic groel protein was protected from proteolysis.
when the same experiment was performed in cells that were depleted for yidc, gp <dig> was not digested suggesting that it was not inserted into the membrane under these conditions.
we conclude, that gp <dig> uses the yidc-only pathway for insertion similar to gp <dig>  <cit> .
in contrast to our in vivo experiments, earlier in vitro data with artificial liposomes consisting of dopc and dopg had suggested that the gp <dig> protein inserts sponanteously into the membrane  <cit> .
very recently, similar gp <dig> variants to our gp <dig> fusion proteins were described that allowed a display on the phage  <cit> .
in contrast to our work, a phagemid system was used and the n-terminus of gp <dig> fusion protein had a pelb signal sequence attached.
this likely changes the route of membrane insertion to the sec-translocase and allows the translocation of large n-terminal domains across the cytoplasmic membrane.
compared to the phagemid system used in previous reports  <cit> , we present a new method of gp <dig> phage display which allows a polyvalent phage display without the need of an n-terminal signal sequence and helper phage infection.
in our system the only gp <dig> copy available is the modified gp <dig> protein on a plasmid when amber  <dig> phage was used.
therefore, all gp <dig> proteins on the phage particle possess the modified n-terminus.
further, our system allows to clearly determine the extend of interference of the modified protein with the propagation cycle of the phage.
when no interference is observed and the gp <dig> fusion protein can substitute for the wild-type, amber  <dig> phage can be used to generate modified phage particles without a modification of the phage genome.
this application might be useful for systems that are sensitive to genetically modified organisms according to -rules.
CONCLUSIONS
bacteriophage m <dig> is suitable for phage display not only with a modified gp <dig> but also with a modified gp <dig> which is a minor coat protein at the phage tip.
the modified gp <dig> protein can be supplied in trans from a plasmid and fully complements an amber  <dig> phage mutant.
the modified phage tip is very well accessible to specific antibodies.

 the bacteriophage m is assembled during a secretion process in the cytoplasmic membrane of escherichia coli. membrane inserted phage proteins contact the single stranded phage dna in an helical array and pass through the outer membrane by a porin-like structure composed of gp. in the inner membrane a protein complex probably consisting of gp gp and thioredoxin catalyses the assembly process. first, membrane inserted gp and gp proteins form a tip structure that is extended by a multiple array of gp proteins, the major coat protein. gp is synthesised as a precursor protein, termed procoat, that is inserted into the inner membrane by the yidc protein and is then processed by leader peptidase. after processing, the transmembrane coat proteins assemble into oligomers and bind to the viral dna forming the nascent phage filament. this filament traverses the outer membrane through the gp complex. finally, the membrane inserted gp and gp proteins are assembled onto the extruding phage at the proximal end of the virion terminating phage assembly. the gp protein has been extensively used for the phage display technology. since gp is engaged in the adsorption of the phage onto the host cell certain restrictions on the infectivity of the modified phage have to be encountered. this might be different for gp modifications since this protein is localized at the distal end of the filamentous phage particle. previously, it has been shown that gp is accessible in the phage particle. therefore, gp might be a good target for phage display technology. in addition, an attractive idea is to have both ends supplied with functional peptide moieties applicable as molecular measures or bifunctional binders. gp is a amino acid long protein that is synthesised without a signal sequence. it is thought that the membrane-inserted protein displays its n-terminus into the periplasm. however, the first amino-terminal residues are hydrophobic and it is questionable whether the protein spans the entire bilayer. one possibility to explore this is to fuse hydrophilic peptides onto the n-terminus. when these modified gp proteins are inserted into the membrane their amino-terminal region can be analysed whether they are exposed in the periplasm. therefore, we have fused short antigenic peptides to the n-terminus of gp between the residues and they extend the protein by to amino acid residues. the proteins are inserted into the membrane and efficiently assemble onto phage progeny particles since they can substitute for the wild-type protein. also, the antigenic epitopes are detectable with gold-labelled antibodies by electron microscopy.  antigenic epitopes at the n-terminus of m gp9 to study the assembly of m gp genetic variants were constructed that extend the n-terminal region of the protein with antigenic epitopes. two antigenic peptides, a amino acid residues long t7-tag and a residues long ha-tag were fused to the n-terminus of gp respectively. this was performed on a gene copy on a pms plasmid using a unique muni restriction site that was engineered between the codons and to generate gp9muni. into this site, dna fragments encoding the tag sequences were introduced. in addition, longer fragments were introduced which encode two copies of the antigenic tag sequences, resulting in additional and residues in pms-g9-dt and pms-g9-dha, respectively. then, the functionality of the modified proteins was tested by complementation of an m13am phage infection. e. coli k bearing the corresponding plasmid was grown overnight in lb medium and plated with top agar containing mm iptg. after solidification of the top agar μl of a phage suspension was applied on top of the agar. plaque formation was observed after incubation at 37°c overnight. when the cells with pms-g9-ha were infected with m13am clear plaques with a turbid zone were visible on the bacterial lawn. whereas no plaques appeared with the k cells containing the pms plasmid, pms-g7/ transformed cells showed plaque formation down to the 105-fold dilution step. in the absence of iptg plaque formation was observed at the 104-fold dilution which is most likely due to a low expression or to recombination events. when k cells with the pms-g9-t or with pms-g9-ha were used plaque formation was evident down to the 105-fold dilution step. similarly, the plasmids encoding the double tags showed efficient plaque formation, as it was observed on the plates with the suppressor containing e. coli k cells. these results suggest that the gp variants expressing the epitope-tagged proteins are functional and allow normal phage propagation. expression of the modified gp protein in e. coli the plasmid-encoded gp variants were analysed for expression in e. coli k the cells were grown at 37°c to the early exponential phase in m minimal medium. protein expression was induced by adding mm iptg and after min the newly synthesised proteins were pulse-labelled for min with 35s-methionine. the total bacterial proteins were tca precipitated to remove the non-incorporated 35s-methionine and immunoprecipitated using an antiserum to the t tag or to the ha tag, respectively. since gp is a very small protein of amino acids containing only two methionines the protein band on a sds tricine page is difficult to visualise. when comparing the protein pattern of cells expressing gp9-t with cells containing the empty plasmid a protein band of about kda was observed. also a weak band of gp9-ha was visible on the gel. the size of the protein was estimated in relation of the major coat protein gp shown in lane since the amino acid residues long gp has a molecular weight of kda, the gp9-t with residues and gp9-ha with residues are proteins of very similar molecular weight. membrane insertion of gp9-t7 the membrane insertion of gp with the n-terminal t tag was analysed in e. coli k cells bearing the pms-g9-t plasmid. the gp9-t protein was expressed as described above. the cells were converted to spheroplasts and analysed by protease mapping. the protein immunoprecipitated with antiserum to the t tag was readily digested by proteinase k added to the outside of the spheroplasts. this suggests that the antigenic tag of gp was accessible to the protease at the periplasmic surface, whereas the cytoplasmic groel protein was protected from digestion. further, the periplasmic portion of the ompa protein was digested by the proteinase k confirming the proteolytic activity. the insertion of gp9-t into the membrane was then investigated in e. coli js in these cells, the membrane insertase yidc can be depleted when the cells are grown in the presence of glucose. after h growth under glucose conditions the cells were pulse-labelled with 35s-methionine for min and converted to spheroplasts. the protease mapping shows that the yidc depleted cells did not allow the digestion of the t7-epitope at the n-terminus of gp. these results suggest that the membrane insertion of gp9-t is yidc-dependent. in both cases, the integrity of the spheroplasts was verified by the protection of groel and the proteolytic activity was corroborated by the accessibility of the ompa protein. assembly of gp variant proteins onto phage assembly of the plasmid-encoded variants onto phage was first followed by dot-blot analysis of phage particles. m13am infections in e. coli k bearing a plasmid coding for one of the gp variants were performed and the progeny phage were collected and titrated. equal amounts of phage was applied on nitrocellulose, incubated with antiserum to m gp to t tag or to the ha tag, respectively. the reaction with a secondary peroxidase coupled antibody was analysed by chemoluminescence. whereas the infecting m13am phage reacted only to the anti gp serum, the phage grown in cells with pms-g9-t clearly reacted with the t serum. similarly, phage from cells expressing the double tag gp9-dt also reacted with the serum to the t tag. strong signals were obtained with gp proteins with the ha epitopes whereas the uninfected k cells expressing gp9-t or gp9-ha showed only a low signal in the corresponding supernatants. this verifies that the plasmid encoded gp proteins with the epitope tags were efficiently assembled onto the phage particles. the exposure of the antigenic epitopes on the phage particles was then tested with immunogold. first, phage was incubated with the respective serum, then with protein a coupled immunogold particles and applied to coated copper grids. after staining with 5% phosphotungstic acid the phage particles were inspected. several gold nanoparticles were bound to the tip of individual phages either with the t tag the double t tag, or the double ha tag. the parental m13am phage, used as a control showed no binding of the gold nanoparticles to the tip. in contrast, for both complemented phage particles we found that about 30% of the gold nanoparticles were bound to phage particles and about 20% of the phage had a gold nanoparticle bound at the tip. taken together, the analysis shows that the modified gp proteins are well exposed and accessible to antibodies. discussion the minor coat protein gp of the filamentous phage m is exposed from the phage particle and can be modified with short peptides. here we have shown that peptides of and amino acid residues can be incorporated into the amino-terminal region of the protein without interfering with membrane insertion and assembly of the phage. the epitopes of these peptides are accessible by antibodies and allow binding of gold nanoparticles that can be visualised by electron microscopy. this implicates that gp could be used for the phage display methodology allowing a combination with the well-established display of modified gp previous experiments have shown that gp of the closely related fd phage is localised at the distal end, together with gp. in that study, it was also shown, that the gp protein is exposed to the surface in contrast to gp however, a fusion with glutathione-s-transferase did not support phage production suggesting that membrane insertion or phage assembly is inhibited. since gst is a folded structure of about kda we tested smaller fusion proteins that may be tolerated for membrane insertion and phage assembly. by introducing short antigenic sequences between the amino acid residues and of gp on a plasmid membrane insertion and phage assembly was followed. also, longer fusions consisting of and additional residues that code for two tandem tags were constructed. intriguingly, all gp fusion proteins complement an amber- phage infection and lead to progeny production up to wild-type levels. when the phage progeny particles were analysed for the presentation of their antigenic epitopes we observed by dot-blot analysis and immunogold labelling a clearly positive response. we conclude that the amino-terminal end of gp is capable to accept modifications and provides a new possibility for phage display. the extended amino-terminal region with an antigenic tag allowed the investigation of the membrane insertion of gp in detail. previously, it had been shown by ftir spectroscopy that the membrane-inserted protein has a high α-helical conformation and adopts a transmembrane conformation. in a short pulse, the synthesised gp was radioactively labelled and analysed for membrane insertion by protease added to the outside of the membrane. indeed, the protease removed the antigenic tag at the n-terminus of gp whereas the cytoplasmic groel protein was protected from proteolysis. when the same experiment was performed in cells that were depleted for yidc, gp was not digested suggesting that it was not inserted into the membrane under these conditions. we conclude, that gp uses the yidc-only pathway for insertion similar to gp. in contrast to our in vivo experiments, earlier in vitro data with artificial liposomes consisting of dopc and dopg had suggested that the gp protein inserts sponanteously into the membrane. very recently, similar gp variants to our gp fusion proteins were described that allowed a display on the phage. in contrast to our work, a phagemid system was used and the n-terminus of gp fusion protein had a pelb signal sequence attached. this likely changes the route of membrane insertion to the sec-translocase and allows the translocation of large n-terminal domains across the cytoplasmic membrane. compared to the phagemid system used in previous reports, we present a new method of gp phage display which allows a polyvalent phage display without the need of an n-terminal signal sequence and helper phage infection. in our system the only gp copy available is the modified gp protein on a plasmid when amber phage was used. therefore, all gp proteins on the phage particle possess the modified n-terminus. further, our system allows to clearly determine the extend of interference of the modified protein with the propagation cycle of the phage. when no interference is observed and the gp fusion protein can substitute for the wild-type, amber phage can be used to generate modified phage particles without a modification of the phage genome. this application might be useful for systems that are sensitive to genetically modified organisms according to -rules.  bacteriophage m is suitable for phage display not only with a modified gp but also with a modified gp which is a minor coat protein at the phage tip. the modified gp protein can be supplied in trans from a plasmid and fully complements an amber phage mutant. the modified phage tip is very well accessible to specific antibodies. 
the amino-terminal region of gp was modified by inserting short sequences that encode antigenic epitopes. the exposure of the antigenic tags on the phage was visualised with immunogold labelling by electron microscopy and verified by dot blotting with antibodies to the tags. we show here that the modified gp proteins correctly integrate into the membrane using the membrane insertase yidc exposing the modified epitope into the periplasm. filamentous m phage extrude from infected escherichia coli with a tip structure composed of gp and gp this tip structure is extended by the assembly of the filament composed of the major coat protein gp finally, gp and gp terminate the phage structure at the proximal end. up to now, gp has been the primary tool for phage display technology. however, gp gp and gp could also be used for phage display and these phage particles should bind to two different or more surfaces when the modified coat proteins are combined. our results suggest that gp at the phage tip is suitable for the phage display technology. therefore, we tested here if the amino-terminal end of gp can be modified and whether the modified portion is exposed and detectable on the m phage particles. the proteins are then efficiently assembled onto the phage particles. the modified gp can be supplied in trans from a plasmid and fully complements m phage with an amber mutation in gene the modified phage tip is very well accessible to antibodies. also extensions up to amino acid residues at the amino-terminal end of gp did not interfere with membrane integration and phage assembly.
 filamentous m phage extrude from infected escherichia coli with a tip structure composed of gp and gp this tip structure is extended by the assembly of the filament composed of the major coat protein gp finally, gp and gp terminate the phage structure at the proximal end. up to now, gp has been the primary tool for phage display technology. however, gp gp and gp could also be used for phage display and these phage particles should bind to two different or more surfaces when the modified coat proteins are combined. therefore, we tested here if the amino-terminal end of gp can be modified and whether the modified portion is exposed and detectable on the m phage particles.  the amino-terminal region of gp was modified by inserting short sequences that encode antigenic epitopes. we show here that the modified gp proteins correctly integrate into the membrane using the membrane insertase yidc exposing the modified epitope into the periplasm. the proteins are then efficiently assembled onto the phage particles. also extensions up to amino acid residues at the amino-terminal end of gp did not interfere with membrane integration and phage assembly. the exposure of the antigenic tags on the phage was visualised with immunogold labelling by electron microscopy and verified by dot blotting with antibodies to the tags.  our results suggest that gp at the phage tip is suitable for the phage display technology. the modified gp can be supplied in trans from a plasmid and fully complements m phage with an amber mutation in gene the modified phage tip is very well accessible to antibodies. 