Patent Application: US-77605997-A

Abstract:
the invention relates to nucleic acid molecules encoding preproproteins having after maturation the biological activity of the mistletoe lectin dimer , to vectors comprising these nucleic acid molecules , to hosts transformed with said vectors and to polypeptides and / or polypeptide dimers which are encoded by these nucleic acid molecules . the polypeptides and / or polypeptide dimers of the invention are widely therapeutically applicable . thus , the present invention further relates to immunotoxins as well as to pharmaceutical compositions that contain the polypeptides and / or the polypeptide dimers of the invention . additionally , the invention relates to diagnostic compositions comprising the nucleic acid molecules of the invention , the polypeptides and / or the polypeptide dimers of the invention and / or primers which hybridize specifically to the nucleic acid molecules of the invention . finally , the invention relates to plant protective agents comprising the polypeptides of the invention and / or the polypeptide dimers of the invention .

Description:
the relative molecular weights were determined by sds polyacrylamide gel electrophoresis under reducing conditions and subsequent protein staining with silver or coomassie brilliant blue or by immunological staining in a western blot analysis . it was surprisingly found that the recombinant , non - glycosylated mistletoe lectin a chain has a relative molecular weight of 25 kda and thus significantly differs from the naturally occurring mistletoe lectin a chains a 1 with 31 kda and a 2 with 29 kda . this difference in relative molecular weight is particularly surprising since it was assumed in the prior art that the a chain is not glycosylated . the recombinant mistletoe b chain has a relative molecular weight of 31 kda and is hence substantially lighter than the glycosylated , naturally occurring mistletoe lectin b chain with 36 kda ( fig7 ). the heterogeneity of naturally occurring ml proteins due to glycosylation and / or sequence variations , which becomes apparent in the sds gel as broad band , does not occur in the recombinant species in any of the cases examined . the relative molecular weights of the reassociated rmla / rmlb holoproteins ( rml ) add up to 56 kda as compared to the heavier nml with 65 - 67 kda . rmla turns out to be an isoelectrically homogeneous protein having an isoelectric point of 6 . 8 as compared to highly purified naturally occurring mistletoe lectin a chains which are divided into 4 species with isoelectric points of 5 . 2 ; 5 . 4 ; 5 . 7 and 6 . 2 ( fig8 ). rmlb proves to be an isoelectrically homogeneous protein having an isoelectric point of 5 . 1 as compared to the naturally occurring mistletoe lectin b chain which is divided into at least 2 species with isoelectric points of 7 . 1 and 7 . 3 ( fig8 ). hence , for the naturally occurring ml holoprotein there is a multitude of possible molecule variants and combinations ( fig8 bottom ) while for recombinant mistletoe lectin proteins there is a uniform mobility in the ief chromatofocusing , which reveals the homogeneity of rml vis - à - vis the microheterogeneity of the naturally occurring protein species . when using immunoaffinity purified rmla preparations in a combined transcription / translation assay , translation inhibiting activity could be detected for rmla ( isolated from the soluble expression production fraction ) and rmla ( isolated from insoluble “ inclusion body ” fraction ). rmla showed a different inhibitory characteristic vis - à - vis the naturally occurring mistletoe lectin a chain with respect to the dosage dependency of the translation inhibition as well as with respect to the non - inhibitable residual translation activity in the reticulocyte lysate used ( see fig9 ). the enzymatic property that forms the basis for the toxic effect of ml holoproteins is significantly reduced in recombinant species . rmlb chains that are produced by renaturation and reoxidation from the primary expression products , like the in vitro reassociated rmla / rmlb , rmla / mlb and mla / rmlb holoproteins have carbohydrate - binding activity that can be detected by enzyme - linked lectin assay ( ella ) by binding to carbohydrate matrices asialofetuin or fetuin . carbohydrate specificity of the recombinant rmlb chain can be determined and quantified in the ella system under competitive conditions for galactose , β - lactose , n - acetylgalactosamine ( galnac ) and sialic acid ( n - acetyl neuraminic acid , nana ). the competitive ella test surprisingly shows different carbohydrate specificities for nmlb and rmlb . the binding affinity is characterized by the system - specific ic50 values for the half - maximal displacement of the proteins of the immobilized asialofetuin ligand by galactose ( ic50 nmlb : 4 . 5 mm ; ic50 rmlb : not determinable due to too low interaction ), β - lactose ( ic50 nmlb : 4 . 9 mm ; ic50 rmlb :& gt ; 70 mm ), n - acetyl - galactosamine ( ic50 nmlb : 20 . 7 mm ; ic50 rmlb : 109 mm ) or of the immobilized fetuin ligand by sialic acid ( ic50 nmlb : 49 . 8 mm ; ic50 rmlb : 47 . 1 mm ). while the nmlb chain described as galactose - specific lectin can be displaced by galactose and β - lactose as expected , the rmlb chain obtained recombinantly in e . coli does not show any detectable interaction with galactose and only poor interaction with β - lactose . recombinant rmlb in turn possesses clear affinity to n - acetylgalactosamine and sialic acid and surprisingly shows a substantial shift of the carbohydrate specificity towards an n - acetyl - galactosamine / sialic acid specific lectin vis - à - vis plant nmlb . with respect to the biological activity of rmlb and rmlb containing holoproteins this results in the possibility of a range of ligands , receptors or target cells that is extended beyond or different from that of plant mistletoe lectin proteins . in a preferred embodiment , the polypeptide according to the invention has at least one chemical or enzymatic modification . this modification can change , reduce or increase the biological activity of the polypeptide , if any . such a modification can be performed , e . g ., after translation and isolation of the polypeptide . such modifications can be also introduced during chemical or semi - synthetical preparation of the polypeptide according to the invention . these modifications can be introduced by the skilled person by methods known per se to alter the pharmacological activity of the mistletoe lectin , preferably to improve it . in another preferred embodiment , the polypeptide according to the invention is a fusion protein . the fusion protein preferably has the above - defined biological activity . this embodiment of the polypeptide according to the invention is also preferably designed to alter the pharmacological properties of the mistletoe lectin polypeptides for targets on the cellular level and preferably to improve them . the invention furthermore relates to a polypeptide dimer having the biological activities of mistletoe lectin , with the two monomers being encoded by the nucleic acid molecules according to the invention . the term “ biological activity of the mistletoe lectin ” is understood to comprise any biological activity from the specter of the entire biological activities of mistletoe lectin . such a function is , e . g ., the pharmacological effect of mistletoe lectin . plant mistletoe 1 induced cytolysis in numerous tumor cell lines of human and murine origin by apoptotic mechanisms [ janssen , 1993 ]. mistletoe lectin 1 or the b chain alone induced the release of cytokines from peripheral mononuclear cells of healthy , human blood donors [ hajto , 1990 ]. mistletoe lectin 1 induced the secretion of superoxide anions from neutrophilic granulocytes of cancer patients [ timoshenko , 1993 ]. mistletoe lectin 1 induced the expression of the a chain of the interleukin 2 receptor ( cd25 ) and / or the hla dq antigen on pheripheral lymphocytes of healthy , human blood donors [ beuth , 1992 ]. after application of mistletoe lectin 1 in mice , an increase in the number of thymocytes , the number of cytotoxic t - lymphocytes ( lyt - 2 +) and helper t - cells ( l3t4 +) in the thymus and the number of peritoneal macrophages , particularly of those carrying the activation marker mac - 3 , could be observed [ beuth , 1994 ]. the relation of l3t4 +/ lyt2 + in the thymus of the experimental animals was increased . in the peripheral blood of the mice the density of the leukocytes , lymphocytes , monocytes in general and in particular of the lymphocytes , which express the interleukin 2 receptor as activation marker on the cell surface , and the monocytes , which express the activation marker mac3 , was increased after treatment with mistletoe lectin 1 [ beuth , 1994 ]. in the blood of cancer patients mistletoe lectin 1 increased the density of t - lymphocytes ( cd4 +, cd8 +), of the natural killer cells and the b - lymphocytes [ beuth , 1992 ]. furthermore , an increase of the endogeneous opiate mediator β - endorphin in the blood plasma of mamma carcinoma patients could be detected after application of mistletoe lectin 1 [ heiny , 1994 ]. it was furthermore ascertained that mistletoe lectin 1 increases the cytotoxic effect of peripheral , natural killer cells vis - à - vis k - 562 tumor cells and the density of large , granular lymphocytes ( lgl ) in the peripheral blood [ hajto , 1989 ]. an antimetastatic activity of mistletoe lectin 1 on sarcoma cells in mice could also be detected [ beuth , 1991 ]. in another embodiment , the polypeptide dimer according to the invention has the same range of biological activities as the naturally occurring mistletoe lectin dimer . the rml holoproteins were produced using the single - chains that had been recombinantly synthesized in a separate step in vitro starting from folded soluble chains or from denatured rmla and rmlb chains in a co - folding step , wherein rmlb was preferably reassociated with a molar excess of rmla in the presence of a glutathion redox system and partially in the presence of protein disulfide isomerase . the rml holoprotein corresponding to the heterodimer was isolated and purified from the reassociation reaction by affinity chromatography on n - acetylgalactosamine agarose or lactosyl agarose , thereby separating it from free rmla and rmla dimers . in an analogous manner rmla / rmlb ( rml ) and rmla / nmlb heterodimer holoproteins were prepared . the cytotoxic effect as an example of the biological activity of reassociated holoproteins was tested on a human monocyte leukemia cell line ( molt4 ). both b chain ( surface binding ) and a chain ( enzymatic ribosome inactivation ) contribute to the cytotoxic effect observed . an in vitro reassociated rmla / rmlb holoprotein as well as an in vitro reassociated rmla / nmlb holoprotein were compared with two batches of naturally occurring nml holoprotein . the recombinant rmla / rmlb and rmla / nmlb holoproteins show comparably high cytotoxic properties with ic values of about 10 - 30 pg / ml ( fig1 ), which reveals the functional integrity and biological activity of the rml holoproteins which were reassociated in vitro using recombinant chains . for the cytotoxic activity to take effect it is necessary that the b chain is operably linked with an enzymatically active a chain , since the isolated rmlb chain alone surprisingly does not have cytotoxic activity . the cytotoxic activity of plant mistletoe lectin b chain preparations which has so far been discussed can therefore probably be attributed to a residual content of nml holoprotein . the recombinant production of the single - chains hence for the first time ever makes it possible to separately describe and employ carbohydrate - binding and enzymatic activity of the mistletoe lectin . the capability for induction of apoptosis as an example of a biological activity of mistletoe lectin was demonstrated for the recombinant rml holoprotein using the monocyte cell line u937 . 24 hrs after treatment of the cells with 70 pg / ml , induction of apoptosis by rml holoprotein could be detected ( fig1 ). tests with mistletoe lectin on molt - 4 cells and peripheral blood mononuclear cells ( pbmc ) showed that in the low dosage range the induction of apoptotic processes is the basis for the cytotoxic activity of the mistletoe lectin . since cytokine induction occurs in the concentration range of low cytotoxicity ( fig1 , fig1 ), a correlation with the apoptosis - inducing activity is plausible , while in the high dosage range as well as with longer incubation times the apoptosis is superimposed by necrotic effects . a cytoxicity as a result of apoptosis could not be detected when treating the sensitive molt - 4 cells with the recombinant b chain so that the biological activity of the apoptosis induction in the low dosage range can only be attributed to the effect of the holoprotein . the immunostimulating effects as examples of biological activities of recombinant mistletoe lectin holoprotein were exemplarily tested for the induction of a tumor necrosis factor α ( tnf - α ) and interferon γ ( ifn - γ ) release from human mononuclear cells of healthy blood donors ( pbmc model ) as well as for the induction of an interleukin - 1α ( il - 1α ) and interleukin - 6 ( il - 6 ) release in a coculture of human primary keratinocytes and skin fibroblasts ( skin 2 zk1200 model ). in the pbmc model , 3 - 48 ng / ml of recombinant rml holoprotein resulted in a dosage - dependent release of tnf - α and ifn - γ , in the skin 2 model , 0 . 25 - 8 ng / ml of recombinant rml holoprotein resulted in a dosage - dependent release of il - 1α and il - 6 . all cytokines mentioned are relevant stimulating mediators of the human immunological system , which have central functions in the activation of particularly the cellular immune response . in contrast to the state of the art up to now , according to which the immunostimulating activity can be mainly attributed to the lectin activity of the b chain [ hajto et al ., 1990 ], none of the above - mentioned cytokine releases could be induced with the recombinant rmlb chain alone . the immunostimulating activity could only be achieved in the low dosage range by operably linked rml holoprotein . this surprising finding suggests that immunostimulating preparations of the plant rmlb chain still contained traces of nml holoproteins and that the immunostimulating effect which had been attributed to the nmlb chain must be attributed to a residual content of nml . while the processes for preparing nmlb which have been described so far do therefore not allow a quantitative separation of holoprotein , the recombinant realization of the individual mistletoe lectin chains for the first time ever makes it possible to examine and provide homogeneous mistletoe lectin b chain preparations . this allows for the first time to separately describe and employ the biological activities of a and b chain and to distinguish the biological activities of single - chains and operably linked holoprotein . ( vi ) biological activities of the recombinant mistletoe lectin b chain ( rmlb ) as marker for the activation of immunocompetent cells , the induction of the cell surface protein cd69 was tested . cd69 appears as one of the first cell surface antigens after activation of t cells , b cells and particularly of natural killer cells ( nk cells ). cd69 has the function of an activation marker of the above - mentioned immunocompetent cell populations since the cell surface protein is not expressed on resting lymphocytes . furthermore , the inducible cd69 surface protein was proven to have a conducive function for the cytolytic activity of the nk cells and tcrγ / δ t cells [ moretta et al ., 1991 ]. flow cytometry ( facs ) using an anti - cd69 mab was employed to detect in the concentration range of 1 - 100 ng / ml an activation of the mononuclear cells both with respect to the occurrence of cd69 on the cell surface and to the share of cd69 - positive cells . the curve for the dosage - dependency was bell - shaped , which points to the necessity for the interlinkage of cellular receptors via both ligand binding sites of the rmlb chain . a cytotoxic effect on the pbmc examined in this test could not be proven , not even at the highest concentration of 100 ng / ml rmlb . in a preferred embodiment , the polypeptide dimer according to the invention exhibits as at least one of the monomers a chemically or enzymatically modified polypeptide according to the invention or a fusion protein according to the invention . modifications can be used to optimize the potency but also to broaden the possible therapeutic uses by eliminating certain qualities ( e . g ., carbohydrate binding sites of the b chain or glycosidase activity of the a chain ) and thus eliminating possible side - effects . polypeptides having modified properties can also serve as tools for elucidating the mechanisms of action . it can become necessary for certain therapies to reduce the antigenicity and the immunogenicity of the polypeptides and / or to optimize their pharmacokinetic properties , which would be possible by specifically exchanging individual amino acids . the invention furthermore relates to antibodies or fragments thereof or derivatives which specifically bind the polypeptide and / or polypeptide dimer according to the invention . they therefore do not recognize the naturally occurring mistletoe lectin or single - chains thereof . preferably , the antibodies according to the invention bind to epitopes which are masked by the glycosylations of the naturally occurring mistletoe lectins . the antibodies can be monoclonal , polyclonal or ( semi ) synthetic antibodies . the fragments can be , e . g ., fab ′, f ( ab ) 2 or fv fragments . antibody derivatives are also known in the art . the invention furthermore relates to a method for preparing the polypeptide or polypeptide dimer according to the invention , in which method the host according to the invention is cultured under appropriate conditions and the so obtained polypeptide or polypeptide dimer is isolated . the person skilled in the art is familiar with the appropriate conditions for culturing and isolating the host . for example , the polypeptide or polypeptide dimer according to the invention can be exported from the host via an appropriate expression system and can be collected in the medium . on the other hand , the polypeptides or polypeptide dimers can remain in the cell and can be isolated from there . in the following , we will discuss another preferred embodiment of the method according to the invention : in order to isolate rmla e . coli cells transformed with an appropriate expression vector were broken up and the soluble and insoluble cell fractions were separated by centrifugation . an analysis of the cell fractions showed that the recombinant mistletoe lectin a chain is accumulated by 5 - 50 % in soluble form and by 50 - 95 % in form of insoluble protein aggregates (“ inclusion bodies ”) depending on the expression conditions and the expression duration . the occurrence of soluble and insoluble proteins indicates that there are at least two methods for the isolation of rmla , if a refolding or renaturation of the rmla proteins is possible . the rmla aggregated to “ inclusion bodies ” was dissolved after washes of the sediments to remove e . coli proteins [ babbitt et al ., 1990 ] under denaturing conditions and was refolded by 90 - fold dilution in folding buffer ( 50 mm tris - hcl , 2 mm dtt , 1 mm edta , ph 8 . 0 ). this treatment resulted in soluble , folded protein species which , as depicted in fig9 had full enzymatic activity just like the renatured , originally insoluble , denatured rmla species . the renatured rmla can be isolated like the soluble rmla by immunoaffinity chromatography using the specific anti - mla antibody ta5 [ tonevitsky et al ., 1995 ]. the presence of rmlb in soluble form as well as in form of insoluble “ inclusion bodies ” indicates that there are two methods for isolating recombinant mistletoe lectin b chain . in order to isolate the soluble rmlb chain from the strongly reductive environment of the e . coli cytoplasm it is incubated in the presence of a redox system consisting of reduced and oxidized glutathion so as to establish the intrachain disulfide bridges and incubated in the presence of the ligand β - lactose in order to stabilize active folding products . from the folding reaction active , carbohydrate - binding rmlb chain was selectively isolated and / or purified in a one - step process by affinity chromatography on lactosyl agarose or n - acetylgalactosamine agarose . in order to isolate rmlb from insoluble expression product fractions which were present as “ inclusion bodies ” the sediment of the e . coli cell complete cell disruption was washed to remove e . coli proteins [ babbitt et al ., 1990 ] and dissolved under denaturing and reducing conditions . renaturation was carried out by dilution in the presence of a redox system consisting of reduced and oxidized glutathion as well as in the presence of the ligand β - lactose . active carbohydrate - binding rmlb chain was selectively isolated and purified from the renaturation reaction by affinity chromatography on n - acetylgalactosamine agarose or lactosyl agarose . the invention furthermore relates to a pharmaceutical composition comprising the polypeptide according to the invention or the polypeptide dimer according to the invention and / or the immunotoxin according to the invention which will be described below , optionally in admixture with a pharmaceutically acceptable carrier . the polypeptides according to the invention , their associates or modifications lend themselves for manifold applications in the therapy of cancer or infections in analogy to the pharmacological properties known for naturally occurring mistletoe lectin . the immunostimulating effects can be used for tumor therapy by directly and / or indirectly stimulating the body &# 39 ; s own immunological defense and enabling it to more effectively combat the tumor and possible metastases . the same holds true for infections , in particular viral infections . the polypeptides according to the invention can be administered in combination with other immunostimulating agents such as interferons , cytokines or colony - stimulating factors , in order to achieve synergistic effects or to reduce the necessary dose of the combination preparation and thus to reduce its side - effects . in combination with cytostatic agents or radiation therapy the side - effect of leucopenia / myelosuppression can be mitigated or reduced so that the weakening of the immunological system that is brought about by the conventional methods of treatment is reduced . the direct cytotoxic effect of the polypeptides having glycosidase activity results in the apoptosis of tumor cells and can also be used for therapeutical purposes . this principle can be made use of more specifically for the application of immunotoxins if the polypeptides according to the invention are coupled to appropriate antibodies . hence , the invention furthermore relates to immunotoxins that comprise at least one polypeptide or polypeptide dimer according to the invention . for example , active a chain or holoprotein can be coupled to antibodies or fragments thereof by methods of protein chemistry . such coupling processes are known to the person skilled in the art , the corresponding conjugates are useful for many purposes [ vitetta , 1993 ]. alternatively , correspondingly constructed fusion protein constructs can be expressed that contain the antigen - binding domain from , e . g ., antibodies and , in addition to that , cytotoxic fragments of the polypeptide according to the invention . furthermore , the formation of metastases can be prevented if the binding of the tumor cells to other cells is inhibited . the polypeptides according to the invention can be used to prevent such binding making use of competitive lectin binding . the invention furthermore relates to a primer and / or a primer pair that specifically hybridizes to the nucleic acid molecule according to the invention or to the complementary strand thereof . b ) a primer and / or a primer pair that specifically hybridizes to the nucleic acid molecule according to the invention or to the complementary strand thereof ; and / or the diagnostic composition according to the invention containing the primer and / or the primer pair can be used to screen organisms for the presence of a lectin gene so as to find , e . g ., new lectin genes that might encode pharmacologically interesting lectins . the nucleic acid molecule according to the invention contained in the diagnostic composition according to the invention can be used to screen organisms for the presence of such lectin genes by , e . g ., southern blot or northern blot methods . by varying the hybridization stringency related lectin genes can be screened for . the polypeptide ( dimer ) can be used , e . g ., to generate antibodies or antisera which can be used to detect by methods known per se respective ( mistletoe ) lectins in various organisms . finally , the invention relates to plant protective agents containing the polypeptide according to the invention and / or the polypeptide dimer according to the invention . the polypeptides according to the invention , their associates or modifications can be used as plant protective agents according to the function discussed for plant mistletoe lectin . the function of the mistletoe lectin as an anti - viral protection is discussed due to its toxic properties as protective measure of the plant against being eaten as well as due to properties that have an effect on the permeability and constitution of membranes . fig1 ( fig1 a , 1 b , 1 c ) show the construction of the primary amplification oligonucleotides ( rml a1 , rml b1 , b2 , b3 , rml a2 , active site regions , ribosome inactivating proteins ; seq id nos : 1 , 2 , 3 , 4 and 5 ; fig2 shows the results of primary viscum album ml amplification product ; fig3 shows the cloning strategy for obtaining the ml gene ( fragments a , b , c , d , e , f , g , h , i , j ); fig4 ( fig4 a , 4 b , 4 c ) show sequences of the inserts of the expression vectors for the rmla and rmlb and complete ml gene sequences , with amino acid sequences ( seq id nos : 1 , 2 , 3 , 4 and 5 ; fig5 shows the construction scheme for an mla - expression vector , pt7 - mla ; fig6 shows the construction scheme for an mlb - expression vector , pt7 - mlb ; fig7 ( fig7 a , 7 b ) show the results of expression of rmla , rmlb ( sds - page , immunological detection ( western blot )); fig8 shows ief chromatofocusing of rmla and rmlb vs . natural ml ( nml ); fig1 shows induction of apoptosis with u937 cells by rml ; fig1 ( fig1 a , 13 b ) shows the immunostimulating effect of recombinant mistletoe lectin ( rml ) in the pbmc model ( induction of secretion of tnf - α and of ifn - γ by rml ); fig1 ( fig1 a , 14 b ) shows the immunostimulating effect of recombinant mistletoe lectin ( rml ) in the skin 2 model ( induction of secretion of il - 1α and of il - 6 by rml ); and fig1 shows induction of the cell surface marker cd69 in pbmc by rmlb . mistletoe lectin ( ml ) belongs to the class of ribosome - inactivating proteins [ stirpe et al ., 1992 ] which represents a protein family widely common to plants of various taxonomic origin . ml was attributed to the group of the type ii ribosome - inactivating proteins due to the activities of its subunits [ endo et al ., 1988a ]. the obvious approach of screening viscum album cdna and genomic libraries , however , is completely inappropriate for finding the ml gene sequence . despite using various dna probes no ml specific clones could be identified in gene libraries from viscum album poly - a + rna . based on the assumption that the ml gene sequence does not contain introns a pcr strategy was followed . since the n - terminal amino acid sequences were known both for mla and mlb chain [ dietrich et al ., 1992 ; gabius et al ., 1992 ], it appeared possible to amplify the mla coding region using degenerate amplification oligonucleotides which had been derived from known peptides ( fig1 a ). while a useful oligonucleotide of low degree of degeneracy can be derived from the n - terminus of the mla chain ( rmla1 , fig1 b )( seq id no : 1 ) it is not possible to construct corresponding sufficiently specific oligonucleotides from the n - terminus of the mlb chain ( rmlb1 , rmlb2 , rmlb3 , fig1 b )( seq id nos : 3 , 4 and 5 ). therefore , alternative strategies had to be developed that made it possible to derive amplification oligonucleotides from yet unknown ml sequence regions by including protein data of related proteins . an amino acid sequence analysis of type i and type ii ribosome inactivating proteins showed a number of conserved regions having high sequence homology . fig1 c ( seq id no : 28 ) illustrates the high degree of kinship between type i and type ii rip for the active center of ricin . within the sequence motif miseaarf it was discussed with respect to e177 and r180 that they play a part in the enzymatic mechanism [ kim et al ., 1992 ; lord et al ., 1994 ]. it was concluded that at least these two residues could be present in the ml sequence . further structural deliberations with respect to the presence of individual residues — paying particular attention to those having a low degree of degeneracy of the codon usage — resulted in the construction of the amplification oligonucleotide rmla2 . the sequence of this oligonucleotide is depicted in fig1 c ( seq id no : 2 ). high molecular , genomic dna was isolated according to the method of baur et al . [ 1993 ] from fresh viscum album leaves ( host tree populus wilsonii ). for the preparation of ml - gene specific dna fragments by pcr 100 ng genomic dna were used for each amplification reaction . amplification was carried out in a total volume of 50 μl , containing pcr buffer ( 10 mm tris - hcl , 1 . 5 mm mgcl 2 , 50 mm kcl , 0 . 25 mm dntp , ph 8 . 3 ), 78 pmol primer rmla1 ( seq id no : 1 ) and 50 pmol ( reaction 2 ) or 100 pmol ( reaction 1 ) rmla2 ( seq id no : 2 ). pcr was carried out with taq dna polymerase ( 1 . 5 u / reaction ) of boehringer mannheim in a biometra thermocycler . the pcr parameters were : 1 min denaturation at 90 ° c ., 1 min annealing at 50 ° c ., 1 min elongation at 72 ° c . for a total of 30 cycles . the amplification products were analyzed by 5 % polyacrylamide gel electrophoresis and staining with ethidium bromide ( fig2 ). the specific amplification product obtained in reaction 2 having a size of about 500 bp was isolated by gel elution and subjected to cloning in ta vectors . the derivatization of the amplification oligonucleotides used for primary pcr is shown in example 1 ( fig1 a ), the preparation of the primary gene fragment of the viscum album ml gene , referred to in the following as “ a ”, is shown in example 2 ( fig2 ). starting from the sequence of the cloned gene fragment “ a ” and from the assumption that the ml gene does not have any introns it was possible to derive sequence - specific 5 ′ oligonucleotides which allowed an amplification of the fragments “ b ”, “ c ”, “ d ” and “ e ”. while the 3 ′ oligonucleotide for “ c ” was also derived from the dna sequence of “ a ”, the degenerate 3 ′ primer for the amplification of the gene fragments “ b ”, “ d ”, “ e ” and “ g ” had to be constructed by analysis of homologous regions of type i (“ b ”) and type ii (“ d ”, “ e ”, “ g ”) rip proteins . the sequence comparisons within the protein families were again used to infer the presence of individual residues , paying particular attention to those residues having a low degree of degeneracy of the codon usage . particularly the known ricin and abrin cdna and derived protein sequences were used to construct the about 50 ml - specific oligonucleotide combinations . fig3 shows only those gene fragments that could be cloned as specific amplification products and could be subjected to further analysis . starting from other oligonucleotide combinations no ml - specific amplification products could be generated . the preparation of the gene fragments “ f ” ( encoding the mla chain ) and “ g ” ( coding of the mlb chain ” is described in detail in example 5 and example 6 , respectively . for an analysis of the 5 ′ and 3 ′ regions of the translated and untranslated sequence regions of the ml gene the conditions for 5 ′ and 3 ′ race [ frohman et al ., 1988 ] were established which lead to the generation of fragments “ h ”, “ i ” and “ j ”. the amplification of fragment “ j ” by race - pcr is thus an alternative to the preparation of complete mlb gene fragments . the race reactions were carried out using cdna that was prepared from isolated viscum album total rna from mistletoe leaves ( host tree populus wilsonii ) by reverse transcription . the inserts of expression vectors pt7 - mla seq id nos . 1 and 2 and pt7 - mlb seq id nos . 3 and 4 were sequenced by standard procedures employing the “ primer walking ” strategy ( detection of completely overlapping sequence of both strands ) using various ml - specific oligonucleotides ( fig4 a , b ) seq id nos : 30 , 31 , 32 and 33 . the underlined sequence regions refer to restriction sites for the cloning into the pt7 expression vectors . both gene fragments have been modified according to the construction scheme of the expression vectors as described in example 5 and example 6 . fig4 c ( seq id nos : 34 and 35 ) shows the complete ml gene sequence seq id nos : 5 and 6 derived from the above fragments . it comprises also 5 ′ and 3 ′ untranslated regions as well as endopeptide and signal - peptide encoding regions . for heterologous expression the sequence encoding the mistletoe lectin a chain was prepared by specific pcr starting from complex genomic mistletoe dna and was terminally modified . translation control elements as well as recognition sequences of restriction endonucleases were added via the non - complementary regions of the primer oligonucleotides used , thereby allowing cloning and separate expression of the mistletoe lectin a chain on the basis of the genomic prepromistletoe lectin . fig5 b shows the preparation of mla encoding gene fragments by pcr . for an amplification of the mla encoding gene sequence 200 ng genomic viscum album dna , 1 . 5 mm ( reaction 1 ) or 2 . 5 mm ( reaction 2 ) magnesium chloride , 40 pmol each of primer oligonucleotide rml16 and rml17 in pcr buffer ( 10 mm tris - hcl , 50 mm kcl , 0 . 25 mm each of dntp , ph 8 . 3 ) were used in a total volume of 50 μl . pcr was carried out using taq polymerase ( 1 . 5 u / reaction , boehringer mannheim ) by a total of 30 cycles of the temperature profile 1 min denaturation at 94 ° c ., 1 min annealing at 52 ° c ., 1 . 5 min elongation at 72 ° c . the amplification products were analysed by 1 % agarose gel electrophoresis and staining with ethidium bromide ( fig5 b ) and provided for cloning in ta vectors by gel elution . the 5 ′ region of the sequence encoding rmla corresponding to the amino acid residues tyrosine 1 - tyrosine 17 [ dietrich et al ., 1992 ; gabius et al ., 1992 ] was prepared as a synthetic gene fragment by hybridization and cloning of two oligonucleotides and by addition of a translation start codon . in this way , the gene sequence was optimized as regards the codon choice such as described for strongly expressed genes in escherichia coli [ gribskov et al ., 1984 ]. at the 3 ′ end of the synthetic rmla gene fragment as well as at the 5 ′ end of the rmla gene fragment obtained by pcr an ssp i restriction site was introduced by the specific exchange of the tyrosine 17 codon from tac to tat which restriction site allowed fusion of the two rmla gene fragments while obtaining vector pml14 - 17 ( fig5 ). the complete generated sequence encoding rmla was confirmed by dna sequencing ( fig4 a ). for expression of rmla in escherichia coli the gene sequence was isolated from vector pml14 - 17 and was put under the control of the t7 - rna polymerase promoter and a transcription terminator by insertion into expression vector pt7 - 7 . the resulting expression vector pt7 - mla ( fig5 ) was used to transform the e . coli expression strain bl21 . for heterologous expression of the mistletoe lectin b chain the complete sequence encoding mlb was amplified from complex genomic viscum album dna by specific pcr , introducing translation control elements as well as recognition sequences for restriction endonucleases via non - complementary regions of the primer oligonucleotide used . fig6 b shows the preparation of the entire gene fragment completely encoding rmlb by pcr . the amplification of the rmlb encoding dna fragment was carried out in pcr reactions in a total volume of 50 μl pcr buffer ( 10 mm tris - hcl , 50 mm kcl , 1 . 5 mm mgcl 2 , 0 . 25 mm each of dntp , ph 8 . 3 ) with 200 ng genomic viscum album dna and 50 pmol primer oligonucleotide rml25 and 30 pmol ( reaction 1 ) and 10 pmol ( reaction 2 ) of primer oligonucleotide rml26 . pcr was carried out using taq polymerase ( 1 . 5 u / reaction , boehringer mannheim ) by a total of 30 cycles of 1 min denaturation at 94 ° c ., 1 min annealing at 52 ° c . and 1 . 5 min elongation at 72 ° c . the pcr products were analysed by 1 % agarose gel electrophoresis and staining with ethidium bromide . the result was a 0 . 8 kbp pcr product which was isolated by gel elution and provided for cloning in ta vectors . the rmlb encoding gene fragment was put under the control of transcription control elements by insertion into expression vector pt7 - 7 and the expression strain e . coli bl21 was transformed with the resulting expression vector pt7 - mlb . integrity of the pcr - generated , complete sequence encoding rmlb was confirmed by dna sequencing ( fig4 b ). expression , immunological analysis , refolding and in vitro reassociation of rmla and rmlb for an expression of recombinant mistletoe lectin a chain 1000 ml lb amp medium were inoculated into 2 l grooved tissue culture flasks with 5 ml of a stationary grown lb amp preculture of e . coli bl21 / pt7 - mla and cultivated at 37 ° c . under shaking . growth was observed by turbidimetry at 578 nm . gene expression was induced by adding 0 . 5 mm iptg when a cell density of od 578 ≈ 0 . 9 - 1 . 0 was reached . for harvesting , the cells were sedimented 2 hrs after induction by centrifugation at 5 , 000 rpm for 20 min and 4 ° c . in a gs - 3 rotor ( sorvall ) and the culture medium was decanted . starting from 1 l culture volume , a cell mass of 3 - 4 g ( wet weight ) was isolated . cell disruption was carried out using a french - press ( sml instruments ). the cell sediment was resuspended in 20 ml disruption buffer b ( 50 mm tris - hcl , 100 mm nacl , 1 mm edta , 5 mm dtt , 1 mm pmsf , ph 8 . 0 ) and disrupted by 2 french - press steps at 1 , 500 psi . insoluble cell fractions and possible “ inclusion bodies ” were sedimented by subsequent centrifugation at 10 , 000 rpm for 30 min and 4 ° c . in a ss - 34 rotor ( sorvall ) and separated from the soluble e . coli proteins or soluble expression product remaining in the supernatant . for an analysis of the expression equal volumina of the cell disruption fractions were examined by 12 . 5 % sds polyacrylamide gel electrophoresis and staining with coomassie brilliant blue as well as by western blot using the mla specific antiserum ta5 ( fig7 a ). the monoclonal antibodies ta5 [ tonevitsky et al ., 1995 ] were provided by the author . like the other antibodies used in the present invention they can be prepared by standard techniques using the respective immunogen ( in the case of ta5 it is ml - 1 or mla ). for detection of the expression equal volumina of the soluble fractions ( lane 2 , 4 , 6 , 8 ) as well as of the insoluble “ inclusion bodies ” fraction ( lane 1 , 3 , 5 , 7 ) of the e . coli disruption were examined for their rmla content . in order to illustrate the course of the expression , samples before ( lane 1 + 2 ), 2 hrs ( lane 3 + 4 ), 4 hrs ( lane 5 + 6 ) and 6 hrs ( lane 7 + 8 ) after induction of gene expression were used . expression is characterized already 1 hr after induction by the occurrence of an immune - reactive 25 kda expression product corresponding to rmla , whose expression maximum is reached already after 2 hrs after induction . the distribution of rmla over the soluble and insoluble cell discruption fraction 2 hrs after induction is ˜ 50 % each , with a longer expression duration resulting in an increase in formation of insoluble “ inclusion bodies ”. the sediment of the e . coli complete cell disruption was washed twice with 20 ml stet - buffer each ( 50 mm tris - hcl , 8 % ( w / v ) sucrose , 50 mm edta ; 1 . 5 % ( v / v ) triton x - 100 , ph 7 . 4 ) according to babitt et al . [ 1990 ] to remove e . coli proteins . the remaining sediment with the “ inclusion bodies ” contained therein was dissolved in 20 ml denaturation buffer ( 6 m guanidiniumchloride , 100 mm dtt , 50 mm tris - hcl , ph 8 . 0 ) by incubation for 16 hrs at room temperature under constant agitation . for renaturation of rmla the protein solution present in the denaturation buffer was slowly and dropwise added to the 90 - fold volume of folding buffer ( 50 mm tris - hcl , 2 mm dtt , 1 mm edta , ph 8 . 0 ) and incubated for 16 hrs at room temperature while stirring it . precipitated protein was separated by centrifugation at 6 , 000 rpm for 30 min and 4 ° c . in a gs - 3 rotor ( sorvall ). the rmla containing supernatant was adjusted for storage to 20 % ( v / v ) glycerol and stored at 4 ° c . for a 1 - step purification of rmla ( soluble expression product fraction or refolded protein ) by immunoaffinity chromatography 260 μg of the monoclonal antibody anti - nmla - igg ( ta5 , tonevitsky et al ., 1995 ) directed against the mistletoe lectin a chain were covalently immobilized on protein a sepharose cl4b ( sigma , deisenhofen ) according to the method by harlow & amp ; spur [ 1988 ]. after incubation of the immunoaffinity matrix with the rmla sample and after washing the matrix with 10 column bed volumes of washing buffer 1 ( 1 m nacl , 10 mm phosphate buffer , ph 7 . 0 ) and 10 column bed volumes of washing buffer 2 ( 10 mm phosphate buffer , ph 7 . 0 ) to remove unspecifically bound proteins specifically bound rmla was eluted with elution buffer ( 0 . 1 m glycine , ph 2 . 5 ). elution was performed to readjust the ph in a receptacle containing 1 m phosphate buffer , ph 8 . 0 . for an expression of recombinant mistletoe lectin b chain 1000 ml lb amp medium were inoculated into 2 l grooved tissue culture flasks with 5 ml of a stationary grown lb amp preculture of e . coli bl21 / pt7 - mlb and cultivated at 37 ° c . under shaking . growth was observed by turbidimetry at 578 nm . gene expression was induced by adding 0 . 5 mm iptg when a cell density of od 578 ≈ 0 . 9 - 1 . 0 was reached . for harvesting , the cells were sedimented 4 hrs after induction by centrifugation at 5 , 000 rpm for 20 min and 4 ° c . in a gs - 3 rotor ( sorvall ) and the culture medium was decanted . the cells were disrupted using a french - press ™ ( slm instruments ). the cell sediment was resuspended in 20 ml disruption buffer b ( 20 mm phosphate buffer , 50 mm nacl , 1 mm edta , 1 mm pmsf , ph 7 . 2 ) and broken up by 2 french - press steps at 1 , 500 psi . insoluble cell fractions and possible “ inclusion bodies ” were sedimented by subsequent centrifugation at 10 , 000 rpm for 30 min and 4 ° c . in a ss - 34 rotor ( sorvall ) and separated from the soluble e . coli proteins or soluble expression product remaining in the supernatant . for an analysis of the expression equal volumina of the cell disruption fractions were examined by 12 . 5 % sds polyacrylamide gel electrophoresis and staining with coomassie brilliant blue as well as by western blot using the mlb - specific antiserum tb33 ( fig7 b ). the monoclonal antibodies tb33 [ tonevitsky at al ., 1995 ] were provided by the author . they were prepared using standard techniques . corresponding antibodies can be prepared by the person skilled in the art by standard techniques employing ml - 1 or mlb as immunogen . for an analysis of the expression equal volumina of the soluble fraction ( lane 2 , 4 , 6 , 8 ) as well as of the insoluble “ inclusion bodies ” fraction ( lane 1 , 3 , 5 , 7 ) of the e . coli were examined for their rmlb content . in order to illustrate the course of the expression , samples before ( lane 1 + 2 ), 2 hrs ( lane 3 + 4 ), 4 hrs ( lane 5 + 6 ) and 6 hrs ( lane 7 + 8 ) after induction of gene expression were used . expression is characterized already 1 hr after induction by the occurrence of an immune - reactive 31 kda expression product corresponding to rmlb , whose expression maximum is reached after 4 hrs after induction . the distribution of rmlb over the soluble and insoluble cell disruption fraction 4 hrs after induction is ˜ 50 % each , with a longer expression duration resulting in an accumulation of expressed rmlb in the form of insoluble “ inclusion bodies ”. the sediment of the e . coli complete cell disruption was washed twice with 20 ml stet - buffer each ( 50 mm tris - hcl , 8 % ( w / v ) sucrose , 50 mm edta ; 1 . 5 % ( v / v ) triton x - 100 , ph 7 . 4 ) according to babitt et al . [ 1990 ] to remove e . coli proteins . the remaining sediment with the “ inclusion bodies ” contained therein was dissolved in 20 ml denaturation buffer ( 6 m guanidiniumchloride , 100 mm dtt , 50 mm tris - hcl , ph 8 . 0 ) by incubation for 16 hrs at room temperature while shaking it . for renaturation of rmlb the protein solution present in the denaturation buffer was slowly and dropwise added to the 90 - fold volume of folding buffer ( 20 mm phosphate buffer , 50 mm kcl , 1 mm edta , 100 mm glucose , 10 % ( v / v ) glycerol , 10 mm β - lactose , ph 5 . 5 ) and incubated for 16 hrs at room temperature while stirring it . precipitated protein was separated from the soluble , folded rmlg fraction by centrifugation at 6 , 000 rpm for 30 min and 4 ° c . in a gs - 3 rotor ( sorvall ). in order to isolate active , carbohydrate - binding rmlb , an n - acetyl - galactosamine agarose affinity matrix ( pierce , usa ) was equilibrated with 10 column bed volumina chromatography buffer ( 50 mm phosphate buffer , 300 mm nacl , 1 mm edta , 10 % ( v / v ) glycerol , 0 . 05 % ( v / v ) tween ®- 20 , ph 7 . 0 ). the application of the samples was carried out by batch incubation of the affinity matrix in an rmlb - containing sample solution for at least 2 hrs at 4 ° c . after washing the affinity matrix with chromatography buffer to remove unspecifically bound proteins bound rmlb was eluted by competitive displacement with 0 . 3 m n - acetyl - galactosamine in chromatography buffer , ph 4 . 0 . ( vii ) in vitro reassociation of mistletoe lectin chains to prepare the holoprotein recombinant mistletoe lectin holoprotein ( rml ) can be prepared starting from isolated , folded mistletoe lectin a and mistletoe lectin b chains or starting from denatured ones which were renatured by co - folding . for reassociation of isolated , folded single - chains , isolated mistletoe lectin b chain ( nmlb or rmlb ) was incubated with a molar excess of rmla in 20 mm phosphate buffer , 50 mm nacl , 1 mm edta ; ph 7 . 2 at 4 ° c . for 16 - 48 hrs . for the interchain disulfide bridges to form , the reaction was incubated in the presence of a redox system of 6 mm glutathione ( ratio of reduced to oxidized 5 : 1 ) or 10 mm glutathione ( ratio of reduced to oxidized 2 : 1 )+ 1 μm protein disulfide isomerase ( boehringer mannheim ). for reassociation starting from denatured single - chains by co - folding , rmla chain was dissolved in 6 m guanidinium chloride , 2 mm dtt , 50 mm tris - hcl , ph 8 . 0 to a concentration of 2 mg / ml . to completely reduce the cystein residues the rmlb chain was dissolved in 6 m guanidinium chloride , 100 mm dtt ; 50 mm tris - hcl , ph 8 , 0 and rebuffered after incubation at room temperature for 20 min on 6 m guanidinium chloride , 50 mm tris - hcl , ph 8 . 0 by gel permeation on pd - 10 ( pharmacia , sweden ) and adjusted to a concentration of 200 μg / ml . reassociation was carried out by co - folding of rmbl with a molar excess of rmla by slowly diluting the guanidinium solution by 1 : 30 in coupling buffer ( 50 mm sodium phosphate buffer , 50 mm kcl , 1 mm edta ; 10 % ( v / v ) glycerol , 100 mm glucose , 20 mm lactose , ph 8 . 0 ) and incubation at 4 ° c . for 16 hrs . for the interchain disulfide bridges to form , the reaction was incubated in the presence of a redox system of 2 mm glutathione ( ratio of reduced to oxidized 1 : 1 ). the coupling reactions were dialysed against storage buffer ( 50 mm sodium phosphate buffer , 300 mm nacl , 1 mm edta , 10 % ( v / v ) glycerol , 0 . 05 % ( v / v ) tween ®- 20 , ph 8 . 0 ). assay and identification of the heterodimer formed was carried out by sds - page under non - reducing conditions and subsequent western blot analysis using specific , monoclonal antibodies against mistletoe lectin a chain ( ta5 ) or mistletoe lectin b chain ( tb33 ). isolation of the holoprotein formed or separation of free rmla or rmla aggregates was carried out by affinity chromatography on n - acetyl - galactosamine sepharose or lactosyl - agarose as described in ( vi ). 1 - 2 μg rmla , naturally occurring mla , rmlb , naturally occurring mlb or ml holoprotein were focused together with ief protein standards ( biorad , usa ) on servalyt prenets ief - gels ( ph 3 - 10 , 125 × 125 mm , 150 μm , serva heidelberg ). for analysis the proteins were immobilized by semi - dry electroblotting on nitrocellulose membranes ( 0 . 2 μm , schleicher & amp ; schüll , dassel ). immunological staining was performed using a monoclonal mla - specific antibody ( ta5 , tonevitsky et al ., 1995 ) for rmla and nmla or using a monoclonal mlb - specific antibody ( tb33 , tonevitsky et al ., 1995 ) for rmlb , nmlb and ml holoproteins . immune complexes were stained using an anti - mouse igg - igg ( sigma , deisenhofen ) conjugated with alkaline phosphatase and reacting the substrates nbt and bcip ( fig8 ). while highly purified plant mistletoe lectin a chain as well as highly purified mistletoe lectin b chain are isoelectrically inhomogeneous proteins with isoelectric points of the nmla of 5 . 2 : 5 . 4 : 5 . 5 : 6 . 2 and of the nmlb of 7 . 1 : 7 : 3 , the recombinant rmla chain having an isoelectric point of 6 . 8 and the recombinant rmlb chain having an isoelectric point of 5 . 1 is a homogeneous protein ( fig8 ). thus , there is a large heterogeneous number of molecule variants for the naturally occurring ml holoprotein ( fig8 bottom ) while the uniform mobility of recombinant mistletoe lectin proteins reveals the homogeneity of the rml vis - à - vis the microheterogeneity of the plant mistletoe lectins . the protein concentration of rmla ( refolded ) and rmla ( soluble ) purified by immunoaffinity chromatography as well as of naturally occurring mla chain ( nmla ) was determined according to bradford [ 1976 ] using a bsa standard . for the detection and quantification of the enzymatic rrna - n - glycosidase activity of mla a non - radioactive test system was established by using the “ tnt coupled reticulocyte system ” ( promega , usa ). per reaction , equal amounts ( 20 μl ) of the tnt system were preincubated at 30 ° c . for 15 min . for the quantification of the translation inhibition 2 μl of the corresponding buffer were added to the control reactions and 2 μl of gradient mla dilutions ( concentration range 350 - 0 pm ) to the test reactions . from each reaction 2 samples were taken at intervals of 8 min and frozen in liquid nitrogen to stop the reaction . as a measure of the translation activity the relative luciferase amount ( sqrt - cpm ) was determined in a bioluminescence test by a scintillation counter . for each reaction the difference of the sqrt - cpms measured of the samples which were taken at different times was determined as measure of the relative translation activity . the activity in the control reaction without rip was set to 0 % inactivation rate ( iar ). by applying the relative translation inactivation rate against the rmla concentrations used the protein concentration was determined by non - linear regression that results in a 50 % inhibition of translation activity as compared to the control reaction . this ic 50 value is a system - dependent value which allows detection and quantification of the enzymatic activity of rmla ( soluble ), rmlb ( refolded ) vis - à - vis nmla ( fig9 ). fig9 shows the detection of the enzymatic , ribosome - inactivating activity of the recombinant mla chain . both by isolation of the soluble expression product content ( rmla soluble ) and by refolding of the protein ( rmla refolded ) isolated from “ inclusion bodies ” an enzymatically active expression product can be obtained . rmla ( soluble ) and rmla ( refolded ) exhibit corresponding activities with ic 50 values of 10 . 7 ± 1 . 3 pm or 15 . 6 ± 6 . 6 pm . they thus exhibit a lower toxic activity than the naturally occurring mla chain ( ic 50 1 . 1 ± 0 . 7 pm ). detection of the carbohydrate - binding activity of the recombinant mistletoe lectin b chain as well as the comparison of the carbohydrate - binding activities and specificities of recombinant and plant mistletoe lectin b chain is carried out by enzyme linked lectin assay ( ella ) in the presence of competitive carbohydrates . the linkage of the nmlb and rmlb chains was established by using an immobilized asiolofetuin matrix to preponderantly galactose and n - acetyl - galactosamine residues as well as by using an immobilized fetuin matrix to preponderantly sialic acid residues . for an immobilization of the carbohydrate matrix 100 μl of an 1 . 1 mg asiolofetuin ( sigma , deisenhofen ) or 1 . 1 mg fetuin ( sigma , deisenhofen ) solution in 11 ml pbs was transferred to the wells of maxisorp c96 mictrotiter plates ( nunc , wiesbaden ) and incubated at room temperature for 16 hrs . after washing the microtiter plates 3 times with 150 μl / well pbs - t ( 10 mm sodium phosphate buffer , 130 mm nacl , 0 . 1 % ( v / v ) tween ®- 20 , ph 7 . 2 ) the microtiter plates were incubated at 36 ° c . for 1 hr with 200 μl / well pbs - t - 1 % bsa ( 10 mm sodium phosphate buffer , 130 mm nacl , 0 . 1 % ( v / v ) tween ®- 20 , 1 % ( w / v ) bsa , ph 7 . 2 ) to block unspecific binding sites and were then washed as described above . for testing 100 μl b - chain containing preparations were used in a concentration of 100 - 500 ng / ml , preferably of 400 ng / ml . the test concentrations were adjusted by dilutions of the samples in pbs - 0 , 05 % bsa ( 10 mm sodium phosphate buffer , 130 mm nacl , 0 . 05 % ( w / v ) bsa , ph 7 . 2 ). per test concentration and control 2 - 3 replicas were prepared . determination of the control is carried out with pbs - 0 . 05 % bsa or the respective preparation buffer . in order to determine the binding specificities , the samples were incubated in the presence of free , competitive sugars . for a displacement of rmlb , nmlb or ml holoproteins from the binding to the asialofetuin or fetuin matrix , galactose preferably in the concentration range of from 0 - 280 mm , n - acetyl - galactosamine in the concentration range of from 0 - 280 mm , lactose in the concentration range of from 0 - 140 mm or sialic acid in the concentration range of from 0 - 140 mm was used . the plates were incubated at 36 ° c . for 2 hrs after loading and were then washed as described above . to the loaded well 100 μl / well goat anti - mistletoe lectin serum ( 1 : 19800 dilution of the serum pool in pbs - t - 0 . 1 % bsa - tx ( 10 mm sodium phosphate buffer , 130 mm nacl , 0 . 1 % ( v / v ) tween ®- 20 , 0 . 1 ( w / v ) bsa , 1 % ( v / v ) triton ® x100 , ph 7 . 2 ) were added , incubated at 36 ° c . for 2 hrs and then washed as described above . for an assay of the immune complexes per loaded well 100 μl anti - goat igg - igg , conjugated with horseradish peroxidase ( sigma , deisenhofen ) were added to a 1 : 3500 dilution in pbs - 1 % bsa ( 10 mm sodium phosphate buffer , 130 mm nacl , 1 % ( w / v ) bsa ; ph 7 . 2 ) and incubated at 36 ° c . for 1 hr . the wells were then washed 6 times with 150 μl / well pbs - t . for development , 100 μl / well substrate solution ( 1 o - phenylene diamine tablet ( sigma , deisenhofen ) in 25 ml 65 mm citric acid , ph 5 . 0 + 10 μl 30 % hydrogen peroxide ) were added and incubated at room temperature for 20 min in the dark . the reaction was stopped by adding 100 μl / 1 m sulphuric acid / well . evaluation was made by absorption photometry at 450 nm with a reference wavelength of 690 nm and calculation of the ic50 value by description of the measured data by a 4 - parameter fit . fig1 shows the values of the displacement of rmlb and nmlb from the immobilized asiolofetuin ligand by increasing amounts of d - galactose ( fig1 a ), β - lactose ( fig1 b ) or n - acetyl - galactosamine ( fig1 c ) as well as of the displacement of the immobilized fetuin ligands by increasing amounts of sialic acid ( fig1 d ) which were observed in the ella system . the binding characteristics of nmlb and rmlb are described by the ic50 value in relation to the semi - maximal displacement . while the carbohydrate binding of the plant nmlb chain can be mainly competed by galactose ( ic50 = 4 . 5 mm ) and β - lactose ( ic50 = 4 . 9 mm ) the recombinant rmlb chain surprisingly has a clearly altered carbohydrate specificity . in contrast to the nmlb the carbohydrate - binding activity of rmlb cannot be competed by galactose ( ic50 not determinable ) and only to a restricted extent by β - lactose ( ic50 & gt ; 70 mm ). apart from the dramatically reduced affinity vis - à - vis galactose and β - lactose the recombinant rmlb chain has a marked specificity for n - acetyl - galactosamine ( ic50 = 109 mm ). another activity of the binding to sialic acid ligands which was described for nmlb could also be detected for the recombinant mlb chain ( fig1 d ). for the plant nmlb chain ( ic50 = 49 . 8 mm ) and the recombinant rmlb chain ( ic50 = 47 . 1 mm ) identical binding affinities were detected . vis - à - vis the plant , mainly galactose / β - lactose - specific nmlb chain the recombinantly prepared rmlb chain has a clearly distinct carbohydrate specificity which is shifted to direction of the n - acetyl - galactosamine / sialic acid binding . detection of the cytotoxicity of in vitro reassociated rml holoproteins on human lymphatic leukemia cells the integrity of the mistletoe lectin holoprotein was detected by quantitative analysis of the cytotoxicity vis - à - vis the human mononuclear ( lymphatic ) leukemia cell line molt - 4 ( european collection of animal cell cultures no . 85011413 ). molt - 4 cells were cultivated in serum - free mdc - 1 medium ( pan systems , aidenbach ) and adjusted for the test to a cell density of 1 . 5 × 10 5 molt - 4 cells / ml at a viability of & gt ; 98 %. in order to determine the cytotoxicity , per well of a 96 - well microtiter plate 90 μl of a molt - 4 cell suspension corresponding to 18000 cells / well were added and mixed with 10 μl of the sample solution . for the test , mistletoe lectin holoprotein preparations ( batches # 220793 ( madaus ) and brain 12 / 94 which were isolated from mistletoe leaves or mistletoe tea by standard techniques using lactosyl sepharose [ franz et al ., 1977 ]) as well as in vitro reassociated rml holoprotein in a concentration range of 1 - 100 pg / ml were correspondingly used ( 1 . 6 fm - 1 . 66 pm ), with the dilution series being prepared in mdc - 1 cell culture medium . per sample concentration and control 6 replicas were prepared . the cells were incubated in an incubator at 37 ° c . and 5 % co 2 for 72 hrs . quantification of the cytotoxic effect was carried out by determining the viability of the cells using a soluble formazan dye according to the wst - 1 method [ scudiero et al ., 1988 ] using the corresponding cell proliferation reagent wst - 1 ( boehringer mannheim ). the recombinant holoprotein as well as the chimeric holoprotein rmlb / nmlb show identical biological activity vis - à - vis the naturally occurring protein with ic50 values around 10 - 30 pg / ml as regards the molt4 - cytotoxicity . in the tested concentration range ( rmlb up to 1 ng / ml ), however , rmlb does not show cytotoxic activity . induction of apoptosis shown for the example of human monocyte cell line u937 by recombinant mistletoe lectin ( rml ) the detection of the induction of apoptotic processes by recombinant mistletoe lectin ( rmla / rmlb ) is carried out by staining the nucleus with the fluorescent dye dapi [ hotz et al ., 1992 ). the typical apoptotic changes in the nucleus ′ morphology can be made visible under the microscope and can be quantified by counting 500 - 100 cells per sample . it is essential for the sensitivity of the assay to use serum - free medium since the presence of serum proteins dramatically reduces the available amount of lectin ( by about the factor 40 at 10 % fcs , ribereau - gayon et al ., 1995 ). the induction time of 24 hrs allows only partially a direct correlation with the molt assay , since the cytotoxic effect is clearly visible in the viability assay only after 72 hrs , apoptosis , however , is an effect that can be detected earlier . if the incubation times are too long , apoptosis is blotted out by secondary necrosis . fig1 shows a marked increase in the rate of apoptotic u937 cells after treatment with recombinant ml holoprotein . at 70 pg / ml the number of apoptotic cells after 24 hrs in two different cultures in serum - free media is increased by factor 3 . hence , the recombinant mistletoe lectin like the naturally occurring protein [ janssen et al ., 1993 ] is capable of inducing apoptotic cell death . the cytokines tnf - α ( monocytes , macrophages ) and ifn - γ ( t helper cells ) are central mediators within the complex network of the human immunological system . human , mononuclear cells ( pbmc , contain monocytes and lymphocytes ) from healthy blood donors were isolated by ficoll - paque ® density gradient centrifugation in accordance with the instructions of the producer ( pharmacia , sweden ). for induction of the release of tnf - α , the cells ( 4 × 10 6 mononuclear cells / ml ) were incubated in rpmi 1640 medium containing 10 % ( v / v ) fetal calf serum , 100 u / ml penicillin , 100 μg / ml streptomycin first for 18 hrs with the recombinant mistletoe lectin proteins alone and then for further 24 hrs with 1 μg / ml lipopolysaccharides from salmonella abortus equi as costimulating factor at 37 ° c ., 5 % co 2 and & gt ; 95 % relative humidity in u - shaped microtiter cell culture plates in an incubator provided with gas . then the concentration of tnf - α was quantified in the cell - free supernatants by any elisa ( genzyme diagnostics , rüsselsheim ). for induction of the release of ifn - γ , the cells were incubated in the above - mentioned medium for 1 hr with the recombinant mistletoe lectin proteins alone and then for further 65 hrs with 0 . 5 μg / ml phytohemagglutinin - l as costimulating factor as described above . then the respective concentration of ifn - γ was quantified in the cell - free supernatants by an elisa ( endogen inc ., cambridge , usa ). immunostimulating effect of recombinant mistletoe lectin in the skin 2 zk1200 model the skin 2 model established as bioassay consists of a three - dimensional fibroblast - containing skin and a structured epidermis from unkeratinized keratinocytes in their own , naturally secreted matrix [ joller et al ., 1996 ]. the skin tissue pieces ( 11 × 11 mm 2 , prepared from human , primary cells ; advanced tissue sciences inc . ( ats ), la jolla , usa ) were provided on a nylon grid in agarose and transferred to special medium a ( ats , la jolla , usa ) immediately upon receipt . both il - 1α and il - 6 are relevant , stimulating cytokines of the human immunological system . for induction of the release of il - 1α or il - 6 the skin 2 tissue pieces were incubated along with the test substance in 2 ml of special medium b ( ats ; la jolla , usa ) for 24 hrs at 37 ° c ., 5 % co 2 in air and & gt ; 95 % relative humidity in 12 - cup cell culture plates ( corning glass works , corning , usa ). then the concentrations of il - 1α ( quantikine human il - 1α assay , r & amp ; d systems inc ., minneapolis , usa ) and il - 6 ( h - interleukin 6 elisa , boehringer mannheim gmbh ) were quantified in the cell - free supernatants by an elisa . the skin 2 model was characterized by a dosage - dependent release of il - 1α induced by 0 . 25 - 8 ng rml / ml and by a dosage - dependent release of il - 6 induced by 05 - 8 ng rml / ml ( fig1 ). surprisingly , none of the above - mentioned cytokine releases could be achieved with the recombinant rmlb chain alone . this finding absolutely contradicts the prior art knowledge according to which the immunostimulating activity was mainly attributed to the lectin activity of the b chain [ hajto et al ., 1990 ]. activation of immunocompetent cells by recombinant mistletoe lectin b chain ( rmlb ) activation of immunocompetent cells was examined using the induction of the cell surface protein cd69 . cd69 appears as one of the first cell surface antigens after activation of t cells , b cells and particularly of natural killer cells ( nk cells ) which , due to their capability of recognizing and lysing neoplastic cells , play a central part in the natural defense against tumors . cd69 is an activation marker of the above - mentioned immunocompetent cell populations since the cell surface protein is not expressed on resting lymphocytes . furthermore , for the inducible cd69 surface protein a conducive function for the cytolytic activity of the nk cells and tcrγ / δt cells could be detected [ moretta et al ., 1991 ]. for detection of the surface marker on human mononuclear cells by flow cytometry ( facs ) pbmc were isolated by density gradient on a hypaque ( sigma ) similar to example 13 . after dissolving the cells in rpmi 160 medium with 5 % fcs and seeding of about 250000 cells / cup of a microtiter plate the solution was incubated for 4 hrs with 1 , 10 , 30 and 100 ng of the test substance rmlb . incubation for 20 min with fluorescence - labelled anti - cd69 mab in an ice bath was followed by washing with hank &# 39 ; s solution with 5 % fcs . the sedimented labelled cells were added to 200 μl sheath fluid and measured in a facscan ( becton dickinson ). the median fluorescence is applied corresponding to the number of the cd69 cell surface marker per cell as well as the share of the cd69 - positive cells in the entire cell population . in the concentration range of 1 - 100 ng / ml an activation of the mononuclear cells both with respect to the occurrence of cd69 on the cell surface as well as with respect to the share of cd69 - positive cells could be detected . a bell - shaped dosage dependence curve could be observed . a cytotoxic effect on the pbmc examined in the present example could not be detected , not even at the highest concentration of 100 ng / ml rmlb . babbitt , p . c ., west , b . l ., buechter , d . d ., kuntz , i . d . and kenyon , g . l . 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[ 1994 ]: x - ray structure of recombinant ricin a - chain at 1 . 8 å resolution . j . mol . biol . 244 , 410 - 422 . ala leu tyr gly arg thr lys ala asp lys thr ser gly pro lys gln ala leu his gly arg thr lys ala asp lys ala ser gly pro lys gln thr gln leu pro thr leu ala arg ser phe ile ile cys ile gln met ile ser glu ala ala arg phe gln tyr ile glu gly glu met arg thr pro leu glu asp ala ile ser ala leu tyr tyr tyr ser thr cys gly gln ile pro thr leu ala arg ser phe met val cys ile gln met ile ser glu ala ala arg phe gln tyr ile glu gly glu met arg thr arg ala leu thr his ala ile ser phe leu arg ser gly ala ser asn asp glu glu lys ala arg thr leu ile val ile ile gln met ala ser glu arg leu glu asn ser ile val asn ile tyr gly lys ala gly asp val lys asn glu ala arg phe leu leu ile ala ile gln met thr ala glu val ala arg phe arg tyr ile gln asn leu val thr lys asn phe lys asp glu ala arg phe leu leu ile ala ile gln met thr ala glu lys asn glu ala arg phe leu leu ile ala ile gln met 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