Patent Application: US-40007103-A

Abstract:
the present invention relates to a novel allergen from timothy grass pollen , phl p11 as disclosed in seq id no : 2 , and use thereof as a reagent and in a diagnositic kit as well as for immunotherapy .

Description:
fig1 . identification of phl p 11 using sds - page and immunoblot analysis of p . pratense pollen extract . a : pollen extract was reduced and separated by sds - page , followed by staining with coomassie brilliant blue . the faint protein band identified as phl p 11 is marked with an arrow . b : immunoblot analysis of a duplicate gel , where the binding of one patient &# 39 ; s serum ige antibodies is visualized . lane 1 : molecular weight markers , lane 2 : p . pratense pollen extract . fig2 . nucleotide ( seq id no : 1 ) and deduced amino acid sequence ( seq id no : 2 ) of phl p 11 cdna . an open reading frame identical to all clones conformed closely with the codon preference derived from all published p . pratense genes expressed in pollen . the underlined nucleotide sequences represent primers gsp - 1 ( seq id no : 15 ) and gsp - 2 ( seq id no : 20 ). open arrowheads indicate nucleotide differences between the clones that were analyzed ; the standard nucleotide ambiguity codes are used at those positions . homopolymer stretch length variation between the clones is indicated by shading . the sequence shown represents the longest of five analyzed clones , while black arrowheads mark where the other cdnas ended in a poly - a stretch . the amino acid sequence marked by solid underlining represents the 20 residues which were determined by n - terminal microsequencing of the natural pollen protein . a single site for potential n - linked glycosylation is indicated by dotted underlining . fig3 . multiple amino acid sequence alignment of phl p 11 ( seq id no : 2 ) and structurally related proteins . each sequence retrieved from the database is preceded by its accession number : a54002 ( loliurn perenne ; seq id no : 3 ), 1815759 ( phalaris coerulescens ; seq id no : 4 ), s31710 ( oryza sativa ; seq id no : 5 ), p33050 ( zea mays ; seq id no : 6 ), 2765366 ( betula pendula ; seq id no : 7 ), p13447 ( lycopersicon esculentum ; seq id no : 8 ), 2832664 ( seq id no : 9 ) and 398899 ( arabidopsis thaliana ; seq id no : 10 ), s43242 ( seq id no : 11 ) and s43244 ( syringa vulgaris ; seq id no : 12 ), 3256212 ( ligustrurn vulgare ; seq id no : 13 ), and 926885 ( olea europaea ; seq id no : 14 ). all entries are shown in full , except the a . thaliana sequence 2832664 ( seq id no : 15 ) which was truncated to show only the domain aligning with the protein family examined here . positions marked x indicate unidentified or atypical residues . the third through eighth sequence include a putative n - terminal leader peptide . hyphens indicate gaps introduced to maximize the number of aligning residues . fig4 . analysis of recombinant phl p 11 expression in e . coli . an e . coli strain prepared for expression of a mbp - phl p 11 fusion protein was grown to mid log phase and then subjected to a temperature shift in order to de - repress the expression system . samples were prepared by boiling pelleted cells in loading buffer containing sds and β - mercaptoethanol . lane 1 : molecular weight markers , lane 2 : preinduction sample , lane 3 : postinduction harvest , lane 4 : sample of purified protein . proteins were visualized by coomassie brilliant blue staining . fig5 . immunoblot inhibition of ige binding to immobilized p . pratense extract proteins by soluble rphl p 11 . pollen extract was reduced and separated by sds - page , and electroblotted onto nitrocellulose membrane . the membrane was incubated with serum samples from two phl p 11 - sensitized grass pollen - allergic subjects ( a and b ), after preincubation with either bsa ( lane 1 ), rphl p 11 ( lane 2 ) or mbp ( lane 3 ). salts and buffers were purchased from sigma ( st . louis , mo .) and fluka ( buchs , switzerland ). pollen from timothy grass ( phleum pratense ) was obtained from pharmacia allergon ab ( välinge , sweden ). protein analysis by sds - page was performed using 4 - 20 % tris - glycine gels ( novex , san diego , calif .) and for electroblotting hybond - c extra membrane ( amersham life science , amersham , uk ) was used . for immunoblot analysis of ige binding , rabbit anti - ige antiserum ( mlab , uppsala , sweden ) and horseradish peroxidase - conjugated donkey anti - rabbit igg ( amersham life science ) were used , followed by ecl detection ( amersham life science ). preparation of polyadenylated rna from total rna and subsequent synthesis of cdna for rt - pcr were performed using the mrna purification kit and the first - strand cdna synthesis kit , both from amersham pharmacia biotech ( uppsala , sweden ). plasmids pet - 23a (+) and pmal - c2 were purchased from novagen ( madison , wis .) and new england biolabs ( beverly , mass . ), respectively . restriction endonucleases ecori , hindiii , ndei and xhoi , as well as taq dna polymerase and deoxynucleotides were from amersham pharmacia biotech . pfu dna polymerase was purchased from stratagene ( la jolla , calif .). dna from pcr and other enzyme reactions was purified using appropriate wizard kits from promega ( madison , wis .). for solid phase capture of biotinylated pcr products , streptavidin - modified magnetic beads ( m - 280 ) from dynal as ( skøyen , norway ) were used . for large - scale plasmid preparation , the plasmid maxi kit from qiagen ( düisseldorf , germany ) was used . oligonucleotides were obtained from scandinavian gene synthesis ( köping , sweden ). dna sequencing was performed using the t7 sequencing kit from amersham pharmacia biotech and [ α - 35 s ] datp from amersham life science . the e . coli strains used were xl1 - blue mr ( stratagene ) for cloning purposes and bl21 ( novagen ) harboring plasmid pt7pol23 ( 18 ) for expression . hitrap chelating columns ( amersham pharmacia biotech ) were used for immobilized metal ion affinity chromatography ( imac ). buffer exchange and size exclusion chromatography of protein preparations were performed using an fplc system and columns packed with sephadex g - 25 and superdex 75 , respectively ( amersham pharmacia biotech ). quantitative serology for the recombinant allergen was established using pharmacia cap system ( pharmacia diagnostics ), employing reagents and procedures as recommended by the supplier . for ige detection in immunoblot inhibition experiments , an 125 i - labeled anti - human ige antibody from pharmacia diagnostics was used . histamine release from isolated granulocytes of allergic and healthy individuals was measured by a radioimmunoassay ( immunotech , marseille , france ). as a positive control for histamine release capacity of cells , the monoclonal anti - ige antibody e124 . 2 . 8 dε2 ( immunotech ) was used . histamine and sodium chloride solution for skin prick tests were obtained from alk ( hørsholm , denmark ). a total of 188 grass pollen - allergic subjects or serum samples were examined in this study . one hundred and fifty serum samples were from an in - house collection at pharmacia diagnostics , selected on the basis of ige sensitization to p . pratense . thirty - eight subjects were from a vienna clinic and were characterized by case history indicative of grass pollen allergy , positive rast result for timothy grass pollen , and positive skin prick test to grass pollen extract . the allergen sensitization profiles of these subjects were established with natural and recombinant timothy grass pollen allergens as described ( 19 ). serum samples from two non - allergic individuals were included for control purposes . phleum pratense pollen was extracted at room temperature for 2 hrs in 5 ml of distilled water per gram of pollen . after centrifugation for 5 min at 13 000 × g , the clear supernatant was divided into small aliquots and stored at − 20 ° c . until use . the pollen extract was subjected to reducing sds - page and either stained with coomassie brilliant blue or electroblotted onto nitrocellulose membrane . protein blots were blocked for 1 hr at room temperature using either 1 % ( v / v ) tween - 20 in pbs ( 137 mm nacl , 2 . 7 mm kcl , 4 . 3 mm na 2 hpo 4 , 1 . 4 mm kh 2 po 4 ) or 5 % ( w / v ) defatted dry milk in pbs and then incubated overnight with patient serum diluted five - fold in pbs containing 0 . 1 % tween - 20 . after washing in the same buffer , bound ige was visualized using a rabbit anti - ige antiserum followed by horseradish peroxidase - conjugated donkey anti - rabbit igg and ecl detection . an ige - binding protein band corresponding to phl p 11 was identified by immunoblotting using an essentially monoreactive serum sample . the band was excised from a coomassie brilliant blue - stained sds - polyacrylamide gel , homogenized and extracted in 6 m guanidinium hydrochloride . after removal of polyacrylamide fragments by centrifugation , the extracted protein was subjected to 20 cycles of sequencing from the n - terminus using a hewlett - packard g1000a instrument . first - strand cdna was synthesized from purified poly - a + rna using the primer 5 ′- cca gtg agc aga gtg acg agg act cga gct caa gc ( t ) 18 - 3 ′ ( qt ; seq id no : 19 ). for 3 ′- pace , the two nested specific forward primers gsp - 1 ( 5 ′- cat tac ata tgg aca agg gcc csg gct tcg tsg tsa c - 3 ′; seq id no : 15 ) and gsp - 2 ( 5 ′- cat gaa ttc gga cgc gtc tac tgc gac - 3 ′; seq id no : 20 ) were used , together with the two nested universal reverse primers q o ( 5 ′- cca gtg agc aga gtg acg - 3 ′; seq id no : 21 ) and q 1 ( 5 ′- gag gac tcg agc tca agc - 3 ′; seq id no : 22 ). primers gsp - 1 and gsp - 2 were designed from the n - terminal amino acid sequence of the phl p 11 protein while primers q o and q 1 were identical to adjacent parts of cdna synthesis primer qt . first - strand cdna was synthesized from purified poly - a + rna using the primer 5 ′- cca gtg agc aga gtg acg agg act cga gct caa gc ( t ) 18 - 3 ′ ( q t ). for 3 ′- race , the two nested specific forward primers gsp - 1 ( 5 ′- cat tac ata tgg aca agg gcc csg gct tcg tsg tsa c - 3 ′) and gsp - 2 ( 5 ′- cat gaa ttc gga cgc gtc tac tgc gac - 3 ′) were used , together with the two nested universal reverse primers q o ( 5 ′- cca gtg agc aga gtg acg - 3 ′) and q i ( 5 ′- gag gac tcg agc tca agc - 3 ′). primers gsp - 1 and gsp - 2 were designed from the n - terminal amino acid sequence of the phl p 11 protein while primers q o and q i were identical to adjacent parts of cdna synthesis primer q t . to generate a phl p 11 - enriched template for 3 ′- race , second - strand cdna was synthesized by 40 cycles of primer extension of biotinylated gsp - 1 using first - strand cdna as template . the cycling profile used was : 95 ° c ./ 5 min , followed by 40 cycles of 95 ° c ./ 60 sec , 58 ° c ./ 60 sec , 72 ° c ./ 90 sec . the product of this reaction was then immobilized on streptavidin - modified magnetic beads and washed with 0 . 1 m naoh and te ( 10 mm tris - hcl , 1 mm edta , ph 8 . 0 ). in the first round of 3 ′- race , a sample of immobilized second - strand cdna and primers gsp - 1 and q o were used in the cycling profile : 95 ° c ./ 5 min , followed by 40 cycles of 95 ° c ./ 1 min , 72 ° c ./ 2 min . one μl of a 20 - fold dilution of this reaction was used as template together with primers gsp - 2 and q i in the second round of 3 ′- race , with the cycling profile : 95 ° c ./ 5 min , followed by 30 cycles of 95 ° c ./ 60 sec , 58 ° c ./ 60 sec , 72 ° c ./ 90 sec . the gsp - 2 and q i primers were designed to incorporate ecori and hindiii sites , respectively , at the ends of the amplification product . after purification and cleavage with these enzymes , the product was cloned between the ecori and hindiii sites of pbr322 ( 22 ). five candidate clones were subjected to dna sequencing , revealing a single open reading frame corresponding to phl p 11 . amplification of full - length phl p 11 coding sequence from immobilized second - strand cdna was performed using the gsp - 1 primer and the reverse primer pp 11 / r - x ( 5 ′- agt cac tcg agt ggc gtc tcg ggg gcg tc - 3 ′; seq id no : 18 ), which was based on the 3 ′ end of the phl p 11 open reading frame . these two primers were designed to incorporate terminal ndel and xhol sites , respectively , in the pcr product . the then - nocycling profile used in this reaction was the same as that in round two of the 3 ′- race experiment . the amplification product , purified and digested with ndel and xhoi , was cloned between the ndel and xhol sites of a pet - 23a (+)— derivative designed for expression of the gene of interest as a fusion to the maltose binding protein ( mbp ) of e . coil . the resulting full - length construct for expression was verified by dna sequencing . the p . pratense allergen was expressed in e . coli as a fusion to mbp . plasmid dna from one selected clone was introduced into strain bl21 harboring plasmid pt7pol23 which provides t7 rna polymerase in a stringently controlled , temperature - dependent manner ( 18 ). lb medium ( 10 g / l tryptone , 5 g / l yeast extract , 5 g / l nacl , adjusted to ph 7 . 0 using 1 m naoh ) was inoculated 1 : 500 with an overnight culture and first grown at 30 ° c . to mid - log phase . the incubation temperature was then raised to 42 ° c . for 1 hr , followed by 4 hrs at 30 ° c . before harvest . cells were collected by centrifugation at 10 000 × g for 10 min at 4 ° c . and resuspended in 5 ml of buffer a ( 20 mm tris - hcl ph 8 . 0 , 0 . 5 m nacl , 100 mm β - mercaptoethanol , 5 mm imidazole ) per gram ( fresh weight ) of cells . the resuspended cells were ruptured by sonication while kept on ice , followed by centrifugation to remove solid material . following exchange to buffer b ( 20 mm tris - hcl ph 8 . 0 , 0 . 5 m nacl , 5 mm β - mercaptoethanol ) containing 5 mm imidazole using sephadex g - 25 , the supernatant was loaded onto a ni 2 + - charged 5 ml hitrap chelating column for imac . the column was washed with 20 mm imidazole in buffer b and elution was performed with a 20 - 250 mm gradient of imidazole in buffer b . fractions containing the eluted fusion protein were pooled and subjected to a final step of size exclusion chromatography through superdex 75 , equilibrated with non - reducing buffer , to obtain a homogeneous , unaggregated preparation without visible contamination by e . coli proteins . to serve as a negative control in functional studies , mbp alone was expressed from bl21 [ pt7pol23 ] cells harboring the expression vector without insert and the protein purified as above , except that buffer b containing 5 mm imidazole was used in place of buffer a at the stage of cell homogenization . the concentration of mbp - phl p 11 and mbp in the final preparations was determined from their absorbance at 280 run , using calculated extinction coefficients of 1 . 30 and 1 . 47 per mg / ml , respectively . assessment of ige - binding activity of rphl p 11 using pharmacia cap system in vitro ige - binding activity of the purified recombinant allergen was examined in pharmacia cap system , an immunoassay system used for ige antibody detection in clinical diagnosis of atopic allergy . experimental immunocap tests were prepared by covalent immobilization of the purified allergen onto activated cellulose at a concentration chosen to achieve an adequate linear measuring range and a background for negative sera adequately below the conventional cut - off value of 0 . 35 ku a / l . negative control tests carrying mbp alone were prepared using the same protein concentration at immobilization . for determination of specific ige to the whole complement of natural p . pratense pollen proteins , the regular pollen extract - based immunocap test was used . for the purpose of comparison to a previously established recombinant allergen , all serum assays were run in parallel with rphl p 2 immunocap tests . assay controls and calculation of statistical parameters attesting to the quality of the assays were performed using standard assay system routines and software ( pharmacia diagnostics ). the proportion of timothy grass pollen - specific ige directed against rphl p 11 and rphl p 5 was investigated by a rast inhibition - based experiment . serum samples from 10 rphl p 11 - reactive subjects were diluted 1 : 10 in buffer c ( 50 mm sodium phosphate ph 7 . 5 , 0 . 5 % ( v / v ) tween 20 , 0 . 5 % ( w / v ) bsa , 0 . 05 % ( w / v ) nan 3 ) and preadsorbed overnight at 4 ° c . with either rphl p 11 , mbp ( negative control ) or rphl p 5 ( positive control ), all at a final concentration of 10 μg / ml . to ensure conditions of antigen excess on the solid phase , approximately 0 . 2 mg of natural timothy grass pollen protein extract was immobilized to nitrocellulose strips of exactly the same size ( 0 . 6 × 3 cm ). strips were blocked by preincubation with buffer c ( once for 1 hour and twice for 5 minutes ) and then exposed to the preadsorbed sera at 4 ° c . overnight . the following day , strips were washed four times in buffer c and then probed with 125 i - labeled anti - human ige antibody at room temperature overnight . strips were washed again four times in buffer c and dried . the amount of 125 i - labeled anti - human ige antibody was determined using a gamma counter ( wallac , turku , finland ). the percentage inhibition of ige binding after preincubation of sera with rphl p 5 or rphl p 11 was calculated as follows : % inhibition = 100 − 100 ×( cpm rphl p 5 / cpm mbp or rphl p 11 / cpm mbp ). the capacity of the recombinant allergen to bind phl p 11 - specific ige antibodies was studied by ige immunoblot inhibition experiments ( 17 ). sera from two grass pollen allergic subjects with ige reactivity to rphl p 11 were preadsorbed with purified rphl p 11 at 10 μg / ml serum , or , for control purposes , with an equal concentration of mbp or bsa . preadsorbed sera were exposed to nitrocellulose - blotted timothy grass pollen proteins separated by sds - page and bound ige was detected as described ( 17 ). granulocytes were isolated by dextran sedimentation of heparinized blood samples ( 23 , 24 ) from two grass pollen allergic and one non - allergic individuals . aliquots of washed cells were incubated with a range of concentrations ( 0 . 001 μg / ml , 0 . 01 μg / ml , 0 . 1 μg / ml , 1 μg / ml ) of purified rphl p 11 , mbp , and a monoclonal anti - ige antibody . histamine released in the supernatant was measured by radioimmunoassay . total histamine was determined after freeze - thawing of cells . results were displayed as mean values of triplicate determinations and represent the percentage of total histamine . after informed consent was obtained from two grass pollen - allergic and four non - allergic individuals , skin prick tests were performed on their forearms as described ( 25 ). individuals were pricked with 20 μl aliquots of solutions containing different concentrations ( 0 . 1 μg / ml , 1 μg / ml , 10 μg / ml , 100 μg / ml ) of purified rphl p 11 and mbp , and with timothy grass pollen extract , histamine and sodium chloride . the skin reactions were recorded 20 minutes after sample application by photography and by transferring a ballpoint pen - tracing of the wheal area to paper using adhesive tape . mean wheal diameters ( dm ) were determined as follows : dm = 0 . 5 ×( d1 + d2 ) where d1 and d2 represent the largest longitudinal and transverse diameters in mm , respectively . immunochemical detection , isolation and protein sequencing of natural phl p 11 immunoblot analysis of serum from a grass pollen - allergic subject , which lacked ige antibodies to all purified or recombinant allergens from p . pratense currently available ( rphl p 1 , rphl p 2 , nphl p 4 , rphl p 5 , rphl p 6 , rphl p 7 , and rphi p 12 ), revealed predominant igebinding to a single protein band at approximately 20 kda . one faint band in coomassiestained sds - page aligned perfectly with the ige - reactive band in the immunoblot analysis ( fig1 ), although a more abundant protein of slightly smaller size could not unambiguously be ruled out . protein from both these bands was extracted separately and a portion of each applied to nitrocellulose membrane for dot - blot analysis . incubation with the reactive senim and subsequent ige detection allowed a positive identification of the band of slightly higher mw as the target for ige antibodies present in the serum sample ( not shown ). the extracted protein was subjected to n - terminal sequencing and the following 20 - residue determination was obtained : dkgpgfvvtgrvycdpcrag ( residues 1 - 20 of seq id no : 2 ). a database search for homologous sequences revealed an exact match to the rye grass allergen lol p 11 , previously purified and amino acid sequenced by van ree et al . ( 26 ). after back - translation of the n - terminal amino acid sequence into dna , using the codon preference seen in other genes expressed in p . pratense pollen , two nested forward pcr primers ( gsp - 1 and gsp - 2 ) were designed for use in 3 ′- race and rt - pcr . first - strand cdna was synthesized from a poly - a + rna preparation , using a universal oligo - dt primer carrying terminal target sequence for two nested reverse pcr primers , q o and q t , to be used in subsequent steps of amplification . specifically enriched second - strand cdna , generated by 40 cycles of primer extension of gsp - 1 on first - strand cdna , was used as template in the first round of 3 ′- race , carried out with primers gsp - 1 and q o . in a second round , 1 / 1000 of the first round reaction was used as template together with primers gsp - 2 and q i . analysis of this reaction by agarose gel electrophoresis revealed two distinct bands of similar intensity , approximately 700 and 800 bp in size ( not shown ). the use of raised annealing temperature did not change the appearance of the second round 3 ′- race product . the double - band product was therefore tentatively considered genuine and specific . the product was cloned and transformants harboring inserts matching both fragment sizes were identified and analyzed by dna sequencing . all five clones examined contained inserts of nearly identical sequence and it appeared that the difference in size between the two bands seen after the second round of 3 ′- race was due to alternative sites for priming of cdna synthesis ( fig2 ), possibly as a result of heterogeneity in the site of transcript polyadenylation . all clones contained an identical open reading frame with a codon usage that agreed well with that of previously known genes expressed in p . pratense pollen . beyond the observed stop codon , none of the three forward reading frames displayed codons that fulfilled this criterion . in order to obtain a cdna encoding the full - length polypeptide , an rt - pcr reaction was performed using forward primer gsp - 1 and reverse primer pp11 / r - x , the latter designed from the 3 ′ end of the open reading frame . the product of this reaction , which appeared as a single band in agarose gel electrophoresis , was cloned in an expression vector and its sequence confirmed . the open reading frame of the cdna defined a polypeptide of 143 amino acid residues with a calculated isoelectric point of 4 . 8 , a molecular mass of 15 . 8 kda and one potential site for n - linked glycosylation ( fig2 ). a similarity search through the databases available at ncbi ( www . ncbi . nlm . nih . gov ) identified pollen proteins from a range of mono - and dicotyledonous plant species with sequence homology to the polypeptide deduced from the cdna sequence . these included lolium perenne ( rye grass ), phalaris coerulescens ( canary grass ), oryza sativa ( rice ), zea mays ( maize ), betula pendula ( birch ), arabidopsis thaliana , lycopersicon esculentum ( tomato ), olea europaea ( olive ), syringa vulgaris ( lilac ) and ligustrum vulgare ( privet ). the level of amino acid sequence identity within this family of pollen proteins ranged from 32 % to 95 % and an alignment , displaying secondary structure predictions and conserved features , is shown in fig3 . from the sequence comparisons it is clear that the p . pratense allergen is a counterpart of the l . perenne allergen lol p 11 and should therefore be designated phl p 11 . the most prominent difference in primary structure observed between phl p 11 and lol p 11 ( sequence accession no . a54002 ) was a stretch of nine additional amino acid residues (- dlrdapetp ; residues 135 - 143 of seq id no : 2 ) at the c - terminus of phl p 11 , equivalent to a 1 . 0 kda increment in molecular mass . in comparison to the l . perenne homologue , the phl p 11 sequence contained a total of six amino acid substitutions , four of which were non - conservative ( d42n , k56g , d57l , k83t ). at position 103 , which was not determined in the case of lol p 11 , an asparagine . residue was present in the phl p 11 sequence . the two homologues showed conservation of one potential site for n - linked glycosylation ( residue 24 ) and six cysteine residues . as previously shown by van ree et al . ( 26 ), group 11 grass pollen allergens are structurally related to the soybean trypsin inhibitor and may therefore present antigenic structures similar to proteins belonging to this family . very recently , structurally related allergens from english plantain , plantago lanceolata , ( pla 11 ) and goosefoot , chenopodium album , ( che a 1 ) were reported ( 27 , 28 ). the discrepancy between the observed apparent mw of the native phl p 11 allergen by sds - page and the mw calculated from the deduced amino acid sequence is presumably explained by post - translational modification of the native allergen . in support of this is the report by van ree et al . ( 26 ), where the homologous l . perenne protein was shown to carry n - linked glycosylation amounting to approximately 8 % of the total molecular mass , and the conservation of the corresponding glycan attachment site in the amino acid sequence of phl p 11 . expression in escherichia coli and purification of rphl p 11 with the aim of allergenic and serological characterization of the phl p 11 allergen , the protein was expressed in e . coli and purified to homogeneity . because of poor solubility when the allergen was initially expressed with an n - terminal hexahistidine tag as the only engineered addition , we chose instead to produce it as a fusion to the e . coli maltose binding protein as a means to aid solubility . after preparing a construct where transcription of the fusion was under control of the t7 promoter , using e . coli xl1 - blue as a cloning host , the plasmid was transferred to strain bl21 harboring plasmid pt7pol23 . in this binary system the construct is quiescent at 30 ° c . and recombinant protein expression induced by a temperature shift to 42 ° c . ( fig4 ). using this strain for expression , accumulation of mbp - phl p 11 to approximately 10 % of total cellular protein was obtained , as estimated from coomassie - stained sds - page ( fig4 ). analysis of fractionated cellular material revealed that approximately half of the fusion protein was present in the soluble phase ( not shown ). the proportion of soluble protein tended to be higher when the culture had been returned to 30 ° c . after a period of induction at 42 ° c ., as opposed to being kept at 42 ° c . until harvest ( not shown ). in order to minimize aggregation of the soluble fusion protein , post - harvest processing was performed under reducing conditions . after buffer exchange to lower the concentration of reductant in the cleared cell extract , the protein was subjected to a first step of purification by imac . while the eluted material appeared as a single distinct band of the expected size on reducing sds - page , analytical gel filtration indicated the presence of different aggregation forms in addition to the monomer . a step of size exclusion chromatography using superdex 75 was therefore added to the purification process . the final preparation appeared monomeric by analytical gel filtration and free of contaminating bacterial proteins by sds - page . it appeared stable and no formation of aggregates was observed upon storage at − 20 ° c . the final yield of purified protein was 12 mg per liter of bacterial culture , or 1 . 7 mg per gram of cell pellet ( fresh weight ). to examine the ige antibody binding capacity of the recombinant allergen and investigate the frequency and magnitude of phl p 11 - specific ige sensitization among grass pollen - allergics , serological tests were prepared for use in pharmacia cap system . as a control for antibody binding to the mbp part of the fusion protein , tests carrying mbp alone were prepared and used in parallel . upon analysis of serum samples of 184 grass pollen - sensitized subjects using these tests , 59 ( 32 %) of them were found to contain specific ige reactivity to the recombinant allergen ( table ii ). the average level of ige to rphl p 11 in the specifically reactive sera was 16 ku a / l , as compared to 79 ku a / l of ige to natural extract of p . pratense pollen . thus , it appears that on average among these subjects , approximately 20 % of the ige reactivity to p . pratense pollen allergens was directed to rphl p 11 . in two of the sera that showed a positive result with the rphl p 11 test , there was also an apparent binding of ige to mbp alone . for one of these sera the ige determination was in fact higher with the mbp test , and this serum was therefore regarded as lacking ige to rphl p 11 . for the other serum , the contribution by mbp to the ige binding by the fusion protein was only about 1 %, which was considered insignificant . in total , only four sera of all 184 tested ( 2 %) showed detectable ige binding to mbp alone , indicating that mbp may be a suitable fusion partner for recombinant allergen production in instances when a soluble non - fusion protein cannot be efficiently produced in e . coli . for the purpose of comparison , the 184 serum samples were also tested with an assay specific for a previously established major grass pollen allergen , rphl p 2 . ige antibody reactivity directed to this allergen was found in 103 ( 56 %) of all tested subjects , with an average ige level of 11 . 4 ku a / l . binding to rphl p 2 would thereby account for approximately 15 % of the total level of ige to whole , natural extract of p . pratense pollen in this subset of sera , which was 74 ku a / l on average . in summary , the serological analysis shows that the e . coli - expressed rphl p 11 has significant and specific ige antibody binding capacity , comparable in frequency and magnitude to that of rphl p 2 . inhibition of ige binding to natural grass pollen extract by soluble rphl p 11 to compare in a more direct way the ige binding characteristics of recombinant and natural phl p 11 , an immunoblot inhibition experiment was performed . in this analysis , competition for ige binding to immobilized natural allergen by soluble rphl p 11 would be visualized as attenuation of ige binding to immobilized natural phl p 11 after preincubation of patient serum with the recombinant allergen . as a control for unspecific inhibition , both serum samples used were preincubated with bsa and mbp in parallel with the rphl p 11 pretreatment . while the control proteins had no visible effect on ige binding to extract proteins , as compared to preincubation with buffer ( not shown ), pretreatment of the serum samples with rphl p 11 almost completely abolished the autoradiography signal at 20 kda molecular weight ( fig5 ). the result demonstrated that the recombinant protein shared epitopes for human ige antibodies with natural phl p 11 . the contribution of phl p 11 to the total ige binding activity of pollen proteins was further examined by dot blot inhibition experiments in which rphl p 5 , an allergen known for its high ige binding capacity ( 7 ), was used for comparison . equal amounts of pollen protein extract were spotted onto identical pieces of nitrocellulose membrane and exposed to patients &# 39 ; sera that had been preincubated with either rphl p 11 , rphl p 5 or mbp . from serological analyses , these sera were known to contain ige to both phl p 11 and phl p 5 , but not to mbp . as controls , buffer incubation and serum from one non - allergic individual were used . after washing , membrane - bound ige was determined radiometrically and the inhibition effects of rphl p 11 and rphl p 5 were calculated in relation to the mbp - pretreated samples . the results of the experiment are shown in table iii . on average rphl p 11 was found to inhibit 25 % of the ige binding to pollen extract , which corresponds to the quantitative serological data shown in table ii , while rphl p 5 caused an average inhibition of 55 %. we conclude that phl p 11 accounts for a relevant proportion of timothy grass pollen - specific ige antibodies , although smaller than phl p 5 . rphl p 11 induces basophil histamine release and immediate skin reaction in an experiment on basophils from a high - level phl p 11 - sensitized allergic individual , rphl p 11 induced dose - dependent release of histamine , demonstrating its capacity to productively cross - link cell surface - bound ige antibodies . limited histamine release occurred from cells of a low - grade phl p 11 - sensitized subject and none from cells of a non - allergic upon incubation with rphl p 11 . ( table 1 ). evidence of specific biological activity of rphl p 11 in vivo was obtained from skin test experiments . in two sensitized subjects , dose - dependent wheal reactions resulted from challenge with a dilution series of the allergen while no reaction occurred in four non - allergic controls tested . ( table 2 ). the rphl p 11 fusion partner mbp alone gave rise to no reaction in neither these experiments . hence , rphl p 11 exhibited biological activity which fulfilled criteria of specificity . table 2 skin prick tests with rphl p 11 b mbp - rphl p 11 100 10 1 0 . 1 mbp pollen subject μg / ml μg / ml μg / ml μg / ml all conc extract histamine allergic no . 5 . 5 2 . 5 — — — 9 . 0 3 . 6 1 allergic no . 8 . 0 5 . 5 4 . 0 — — 10 . 5 7 . 0 2 non - allergic — — — — — — 8 . 0 no . 1 non - allergic — — — — — — 7 . 5 no . 2 non - allergic — — — — — — 4 . 9 no . 3 non - allergic — — — — — — 8 . 5 no . 4 b two allergics and four non - allergics were skin tested with rphl p 11 at different concentrations , with timothy grass pollen extract and histamine . the diameter of the resulting wheal reactions were determined and are presented in mm . grass pollens belong to the most frequently sensitizing and potent allergen sources . they contain a number of allergenic molecules , several of which have been identified and characterized in the recent past ( 3 ). in the present invention we report the identification , cloning and recombinant production of a novel p . pratense pollen allergen which adds new important epitopes to the growing panel of recombinant grass pollen allergens ( 29 ). despite the fact that the group 11 grass pollen allergens are glycoproteins ( 26 ) and contain several cysteine residues , we were able to produce soluble , monomeric and immunologically active rphl p 11 allergen by utilizing mbp as a fusion partner for expression in e . coli . extensive serological characterization of ige reactivity to rphl p 11 was carried out using a quantitative assay system where allergen is covalently immobilized onto activated cellulose . using the rphl p 11 - specific tests , we found that about one third of all grass pollen sensitized subjects analyzed ( n = 184 ) contained serum ige antibodies binding to rphl p 11 and that the magnitude of binding corresponded to a significant proportion of grass pollen - specific ige antibodies in these subjects . evidence supporting the authenticity of epitope presentation by rphl p 11 was obtained from immunoblot inhibition experiments , where natural grass pollen proteins were attached on solid phase and rphl p 11 used as fluid - phase inhibitor . specific and extensive inhibition of ige binding to the natural allergen occurred in both of two patient sera examined , demonstrating that rphl p 11 could compete with natural phl p 11 for ige antibody binding . taken together , the serological data show that immunoreactive rphl p 11 can be produced using e . coli expression and that the recombinant protein shares epitopes for ige antibodies with the natural allergen . based on the results obtained , it is clear that rphl p 11 represents an important addition to the panel of recombinant grass pollen allergens useful for in vitro diagnosis of grass pollen allergy . relevant to this discussion is the immunological analysis of chemically deglycosylated natural lol p 11 reported by van ree et al . ( 26 ), which suggested the involvement of carbohydrate structures in the ige binding properties of this allergen . thus , we cannot exclude that a qualitative difference in allergenic properties exists between natural phl p 11 and the recombinant molecule described in this paper and that expression of phl p 11 in a glycosylated form , using a eukaryotic host , could yield a recombinant allergen with different ige binding characteristics . on the other hand , in view of recent notions that glycan epitopes may not be efficient elicitors of ige - mediated reactions or informative in relation to clinical allergy manifestation ( 30 - 34 ), it is possible that an unmodified recombinant allergen expressed in e . coli is more useful for diagnostic purposes . despite the significant sequence homology among the members of the widely represented ( grasses , trees and weeds ) group of allergens exemplified by phl p 11 in timothy grass and ole e 1 in olive tree pollen , little cross - reactivity for ige antibodies appears to exist between them . in a preliminary analysis , we have been unable to detect cross - reaction between rphl p 11 and ole e 1 ( niederberger , valenta & amp ; lidholm , unpublished data ) and the results of recent studies on other members of this allergen family ( 27 , 28 ) are in agreement with this observation . one important implication of the apparent lack of significant cross - reactivity between rphl p 11 and other members of this allergen family is that they are useful as diagnostic markers to more precisely identify the primary sensitizer of allergic individuals , as compared to natural extracts or cross - reactive components such as profilin , two - ef - hand allergens or bet v 1 homologues . thus , a preferential ige recognition of phl p 11 , in relation to other members of this allergen family , may suggest a primary sensitization by grass pollen rather than another allergen source containing cross - reactive components . the use of selected recombinant allergens in this way may provide information useful for advice on allergen avoidance and adequate selection of allergen extract for specific immunotherapy treatment . in conclusion , the present invention concerns cdna cloning and recombinant production of an ige - reactive and biologically active group 11 grass pollen allergen . recombinant phl p 11 can be used to identify group 11 allergen sensitization in patients and for specific immunotherapy . 1 . kay , a . b . 1997 . allergy and allergic disease . blackwell science , oxford . 2 . d &# 39 ; 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