the isolation of the cdna clone coding for art v 1a was done in the following way : a cdna expression library in the phage lambda - zap ii ( stratagene , la jolla , calif . ; catalogue no . 237612 ) was prepared . the starting material was mugwort pollen mrna . the cdna library was screened immunologically with a serum pool of 20 patients that had been shown to recognize art v 1 in patient blots . a clone was isolated that reacted positively with the serum pool and on repeated re - screening gave 100 % immunopositive plaques . the immune reaction to this clone was relatively weak but clearly positive . the proof that this clone codes for art v 1 was as done as follows : the major allergen art v 1 was extracted from mugwort pollen under very mild conditions , namely room temperature and extraction with water for 15 – 30 min under light shaking . this extract was further separated by preparative gel electrophoresis and the fractions were tested by immunoblots . fig6 a shows three fractions ( f1 , f2 , and f3 ) that were free of coomassie - stainable protein impurities and contained protein bands migrating at apparent mr between 22 and 29 kda . occasionally , a dimeric band corresponding to about 50 kda is also seen . fig6 b shows the patient blots of these fractions . it is obvious that that the fraction of highest mr binds ige from all three patients tested while the fraction of lowest mr is only weakly recognized by patients &# 39 ; sera . fig6 c was obtained after staining the same blot with the boehringer mannheim glycoprotein detection kit ( dig glycan / protein double labeling kit , catalogue no . 1500783 ). it is clear that all three fractions consist of glycoprotein . all three fractions were analyzed by n - terminal edman degradation and yielded identical n - terminal sequences which are underlined in fig1 – 3 . computer analysis of the deduced protein sequence in fig1 according to nielsen et al . ( 5 ) predicts art v 1 has a typical n - terminal hydrophobic signal sequence that causes targeting to the endoplasmatic reticulum and the golgi apparatus . the sequence after the start of the mature form of the protein predicted by this computer algorithm is identical with the n - terminal sequence found in the natural protein ( underlined in fig1 – 3 ). a very similar n - terminal partial sequence was found previously by matthiesen et al . ( 6 ). it can be concluded that art v 1 is a secreted glycoprotein , whose n - terminus was created by removal of a typical er signal sequence . the natural protein is heterogeneous due to differences in the degree of glycosylation and the fully glycosylated form ( f3 in fig6 ) binds ige best . the primary cdna clone of art v 1 a was used as a hybridization probe after labeling with 32 p by the random priming method . the library described above was screened with this probe and two additional clones were obtained and termed art v 1b and art v 1c . the clones were analyzed by dna sequencing . the sequences are shown in fig2 and 3 . e . coli was used to express the mature ( short ) form of the art v 1a protein as a non - fusion recombinant protein after re - cloning the appropriate part of the cdna in the expression system pmw172 . it is well known that recombinant proteins expressed in e . coli display no postsynthetic modifications ( with the possible exception of n - terminal methionine cleavage ) and in particular , they do not contain sugars . the construction of the expression plasmid is shown in fig7 . the recombinant art v 1a protein was enriched in the soluble fraction of the e . coli proteins . fig8 shows immunoblots of the soluble fraction with 15 individual patients . the apparent mr of the natural protein is about 27 kda ( fig8 , mugwort pollen ) while the apparent mr of the recombinant protein is about 18 kda due to the absence of glycosylation . the theoretical mr is 10 . 8 kda indicating that the recombinant protein shows a very unusual electrophoretic mobility in the sds polyacrylamide gel electrophoresis . it is clearly seen ( fig8 , rart v 1a ) that 10 of the fifteen patients &# 39 ; ige recognize the unglycosylated protein but patients also exist that do not recognize this form of the protein . quite surprisingly , patients 3 , 4 and 10 recognize the unglycosylated protein much better than the natural glycosylated form . the controls in fig8 show that e . coli proteins are not or only weakly recognized by patients and by the secondary antibody . these weak bands can be seen in all three patient blots shown in fig8 . to explain the experimental results presented in the last paragraph , the following hypothesis is put forward . it can be envisioned that the sugar moieties of art v 1 are necessary to bring the protein backbone into its natural conformation so that the ige of some patients can bind the epitopes created in this way . some other epitopes on the other hand can be easily recognized by the patients &# 39 ; sera in the absence of sugar . another less probable explanation for the above observations is that patients &# 39 ; sera that do not recognize the unglycosylated form , in fact recognize epitope ( s ) consisting of sugars . preliminary characterization of the sugar moieties of the natural major allergen of artemisia vulgaris , art v 1 the natural major allergen of artemisia vulgaris was purified to homogeneity by the following methods : 1 . anion exchange chromatography on sepharose q ( pharmacia , uppsala , sweden ) at ph = 5 . 2 and 2 . hplc - gel filtration chromatography on tsk - gel g2000sw ( tosohaas , stuttgart , germany ). the material was homogeneous as judged by n - terminal protein sequence and amino acid analysis , but had a heterogeneous molecular weight due to different glycosylation patterns . the molecular weight was determined by maldi - tof mass spectrometry and showed two broad peaks with a mean molecular mass of 13 . 5 kda and 15 . 5 kda , respectively . the apparent molecular weight determined by sds - page , on the other hand , was 24 to 28 kda , which can be taken as an indication for a very unusual protein structure or for a very unusual structure of the sugar moieties . the preliminary analysis of the sugars covalently - bound to the polypeptide chain by hydrolysis and hplc showed that no n - glycosylation and very likely also no typical 0 - glycosylation are present on the art v 1 allergen . rather , a previously described plant 0 - glycosylation on hydroxyproline residues seems to be the case . art v 1 is a proline - rich protein ( 20 % proline ) and is postsynthetically modified so that in the mature protein proline and hydroxyproline residues are present in a ratio of 4 : 6 . extensive studies with five different lectins ( galanthus nivalis agglutinin , sambucus nigra agglutinin , maackia amurensis agglutinin , peanut agglutinin , and datura stramonium agglutinin ) that are specific for known n - glycans and o - glycans showed that these sugar structures are not present in the art v 1 allergen . the role of this unusual protein structure and unusual sugar moieties on the formation of b - cell epitopes of this major allergen is presently being investigated . fig8 gives the first hint that the sugar moieties might play an important role in the ige recognition of the art v 1 allergen . 1 . charpin , j ., surinyach , r ., and frankland , a . w . ( 1974 ) atlas of european allergenic pollens . sandoz , paris . 2 . spieksma , f . t . m ., charpin , h ., nolard , n ., and stix , e . ( 1980 ) clin . allergy 10 : 319 – 329 . 3 . cornillon , j ., bernard , j - p ., gueho , e ., and touraine , r . ( 1972 ) rev . franc . allergol . 12 : 131 – 135 . 4 . domon , c ., evrard , j . l ., herdenberger , f ., pillay , d . t . n ., and steinmetz , a . ( 1990 ) plant mol . biol . 15 : 643 – 646 . 5 . nielsen , h ., engelbrecht , j ., brunak , s ., and von heijne , g . ( 1997 ) prot . eng . 10 : 1 – 6 . 6 . matthiesen , f ., ipsen , h ., and løwenstein , h . ( 1991 ) in : allergenic pollen and pollinosis in europe . pp 36 – 44 . d &# 39 ; amato , g ., spieksma , f . t . m ., and bonini , s . eds ., blackwell scientific publications , cambridge .
the invention relates to dna molecules which code for the allergen art v 1 or isoforms thereof , the sequence of the allergen , a method for the production of an art v 1 molecule , a vector and a transformed host cell .