Patent Application: US-82881704-A

Abstract:
the invention relates to the production of gonadotrophins and corresponding receptors in transgenic plants . the invention provides a method to produce a gonadotrophin or its corresponding receptor in a transgenic plant with modified glycosylation machinery , in order to allow for mammalian type of glycosidic side chains of gonadotrophin and its corresponding receptor .

Description:
one important enzyme involved in mammalian n - glycan biosynthesis that is not present in plants is β1 , 4 - galactosyltransferase . here , for one , the stable expression of β1 , 4 - galactosyltransferase in tobacco plants is described . the physiology of these plants is not obviously changed by introducing α1 , 4 - galactosyltransferase and the feature is inheritable . crossings of a tobacco plant expressing β1 , 4 - galactosyltransferase with a plant expressing the heavy and light chain of a mouse antibody produced antibody having terminal galactose in similar amounts as hybridoma produced antibodies . herein it is thus shown that the foreign enzyme can be successfully introduced in plants . a clear increase in galactose containing glycoproteins is observed . moreover , this feature is inheritable and there is no visible phenotypical difference between the galactosyltransferase plants and wild type . a mouse monoclonal antibody produced in these plants has a degree of terminal galactoses comparable to hybridoma produced antibody . this shows that not only endogenous proteins become galactosylated but also a recombinantly expressed mammalian protein . a plant transformation vector containing human β1 , 4 - galactosyltransferase was constructed as follows : a 1 . 4 kb bamhi / xbai fragment of pcdnai - galt ( aoki et al ., 1992 ; yamaguchi and fukuda , 1995 ) was ligated in the corresponding sites of puc19 . subsequently , this fragment was re - isolated using surrounding kpni and hincii sites and cloned into the kpni and smai site of prap33 ( named prap33 - hgalt ). using asci and paci sites the camv35s promotor - cdna - nos terminator cassette of prap33 - hgalt was cloned in the binary vector pbinplus ( van engelen et al ., 1995 ). modifications to the published protocol are : after incubation with a . tum ., leaf discs were incubated for three days in medium containing 1 mg / ml of naa and 0 . 2 mg / ml bap and the use of 0 . 25 mg / ml cefotaxime and vancomycine to inhibit bacterial growth in the callus and shoot inducing medium . 25 rooted shoots were transformed from in vitro medium to soil and , after several weeks , leaf material of these plants was analysed . the β1 , 4 - galactosyltransferase rna level in the transgenid plants was analyzed by northern blotting ( sambrook et al ., 1989 ) rna was isolated from leafs of transgenic and control plants as described ( de vries et al ., 1991 ). ten μg of total rna was used per sample . the blot was probed with a [ 32 p ] datp labeled ssti / xhol fragment , containing the whole galt cdna , isolated from pbinplus - hgalt . total protein extracts of tobacco were prepared by grinding leafs in liquid nitrogen . ground material was diluted 10 times in sds page loading buffer ( 20 mm of this - hcl ph 6 . 8 , 6 % glycerol , 0 . 4 % sds , 20 mm dtt , 2 . 5 ìg / ml bromophenol blue ). after incubation at 100 ° c . for 5 min insoluble material was pelleted . supernatants ( 12 . 5 μl / sample ) were run on 10 % sds - page and blotted to nitrocellulose . blots were blocked overnight in 0 . 5 % tween - 20 in tbs and incubated for 2 hours with peroxidase conjugated rca 120 ( ricinus communis agglutinin , sigma ) ( 1 μg / ml ) in tbs - 0 . 1 % tween - 20 . blots were washed 4 times 10 minutes in tbs - 0 . 1 % tween - 20 and incubated with lumi - light western blotting substrate ( roche ) and analysed in a lumianalyst ( roche ). a rabbit polyclonal antibody directed against horseradish peroxidase ( hrp , rockland immunochemicals ) was split in reactivity against the xylose and fucose of complex plant glycans by affinity chromatography with bee venom phospholipase according to ( faye et al ., 1993 ). a rabbit anti lewisa antibody was prepared as described ( fitchette laine et al ., 1997 ). blots were blocked with 2 % milkpowder in tbs and incubated in the same buffer with anti - hrp , anti - xylose , anti - fucose or anti - lewis - a . as secondary antibody alkaline hrp - conjugated sheep - anti - mouse was used and detection was as described above . mgr48 ( smant et al ., 1997 ) is a mouse monoclonal igg that has been expressed in tobacco plants . the construct used for transformation was identical to monoclonal antibody 21c5 expressed in tobacco ( van engelen et al ., 1994 ). flowers of selected tobacco plants with high expression of β1 , 4 - galactosyltransferase were pollinated with plants expressing mgr48 antibody . the f1 generation was seeded and plants were screened for leaf expression of antibody by western blots probed hrp - conjugated sheep - anti - mouse and for galactosyltransferase expression by rca as described above . freshly harvested tobacco leaves were ground in liquid nitrogen . to 50 g of powdered plant material , 250 ml of pbs , containing 10 mm na 2 s 2 o 5 , 0 . 5 mm edta , 0 . 5 mm pmsf and 5 g polyvinylpolypyrrolid , was added . after soaking for 1 hour ( rotating at 4 ° c . ), insoluble material was removed by centrifugation ( 15 min , 15 , 000 g , 4 ° c .). the supernatant was incubated overnight ( rotating at 4 ° c .) with 1 ml of proteing - agarose beads . the beads were collected in a column and washed with 10 volumes of pbs . bound protein was eluted with 0 . 1 m glycine ph 2 . 7 and immediately brought to neutral ph by mixing with 1 m tris ph 9 . 0 ( 50 μl per ml of eluate ). purified antibody was quantified by comparison of the binding of hrp - conjugated sheep - anti - mouse to the heavy chain on a western blot with mgr48 of known concentration purified from hybridoma medium ( smant et al ., 1997 ). hybridoma mgr48 and plant produced mgr48 was run on 10 % sds - page and blotted as described above . detection with rca was as described above . for antibody detection , blots were probed with hrp - conjugated sheep - anti - mouse and detected with lumi - light western blotting substrate as described above . human α1 , 4 - galactosyltransferase ( masri et al ., 1988 ) was introduced in tobacco plants by agrobacterium mediated leaf disk transformation of plasmid pbinplus - hgalt containing a cdna that includes a complete coding sequence . twenty - five plants selected for kanamicin resistance were analysed for mrna levels by northern hybridization ( fig2 a ). the same plants were analyzed by the galactose binding lectin rca 120 ( ricinus cummunis agglutinin ). rca binds to the reaction product of β1 , 4 - galt ( galβ1 , 4glcnac ) but also to other terminal β - linked galactose residues . rca binds to one or more high molecular weight proteins isolated from non transgenic control tobacco plants ( fig2 b ). probably these are arabinogalactan or similar proteins . rca is known to bind to arabinogalactan proteins ( schindler et al ., 1995 ). in a number of the plant transformed with human β1 , 4 - galactosyltransferase , in addition , binding of rca to a smear of proteins is observed . this indicates that in these plants many proteins contain terminal β - linked galactose residues . there is a good correlation between the galactosyltransferase rna expression level and the rca reactivity of the trangenic plants . human β1 , 4 - galactosyltransferase expressed in transgenic plants is therefor able to galactosylate endogenous glycoproteins in tobacco plants . as it is known that galactosylated n - glycans are poor acceptors for plant xylosyl - and fucosyltransferase ( johnson and chrispeels , 1987 ), the influence of expression of β1 , 4 - galactosyltransferase on the occurrence of the xylose and fucose epitope was investigated by specific antibodies . a polyclonal rabbit anti - hrp antibody that reacts with both the xylose and fucose epitope shows a clear difference in binding to isolated protein from both control and transgenic plants ( fig3 ). the effect of expression of β1 , 4 - galactosyltransferase on a recombinantly expressed protein was investigated . three tobacco plants expressing β1 , 4 - galactosyltransferase ( no . galt6 , galt8 and galt15 from fig2 ) were selected to cross with a tobacco plant expressing a mouse monoclonal antibody . this plant , expressing monoclonal mgr48 ( smant et al ., 1997 ), was previously generated in our laboratory . flowers of the three plants were pollinated with mgr48 . of the f1 generation 12 progeny plants of each crossing were analysed for the expression of both antibody and β1 , 4 - galactosyltransferase by the method described in materials and methods . of crossing galt6xmgr48 and galt15xmgr48 no plants were found with both mgr48 and galt expression . several were found in crossing galt8xmgr48 . two of these plants ( no . 11 and 12 ), were selected for further analysis . using proteing affinity , antibody was isolated from tobacco plants expressing mgr48 and from the two selected plants expressing both mgr48 and β1 , 4 - galactosyltransferase . equal amounts of isolated antibody was run on a protein gel and blotted . the binding of sheep - anti - mouse - igg and rca to mgr48 from hybridoma cells , tobacco and crossings galt8xmgr48 - 11 and 12 was compared ( fig4 ). sheep - anti - mouse - igg bound to both heavy and light chain of all four antibodies isolated . rca , in contrast , bound to hybridoma and galt plant produced antibody but not to the antibody produced in plants expressing only mgr48 . when the binding of sheep - anti - mouse - igg and rca to the heavy chain of the antibody is quantified , the relative reaction of rca ( rca binding / sheep - anti - mouse - igg binding ) to galt8xmgr48 - 11 and 12 is respectively 1 . 27 and 1 . 63 times higher than the ratio of hybridoma produced antibody . this shows that rca binding to the glycans of antibody produced in galt plants is even higher than to hybridoma produced antibody . although the galactosylation mgr48 from hybridoma is not quantified , this is a strong indication that the galactosylation of antibody produced in these plants is very efficient . there is a need for an accessible and standardised source of fsh for therapeutic and diagnostic purposes , which is guaranteed to be free of lh activity . fsh preparations normally are derived from ovine or porcine pituitaries , which always implies the presence of ( traces of ) lh , and the risk of contamination with prion - like proteins . substitution of brain derived fsh for plant produced recombinant fsh may be a good method of eliminating these problems . furthermore application of plant produced fsh receptor ( fshr ) in a diagnostic testkit provides a good method for measurement of bioactive fsh by receptor assay . however , production of bioactive animal glycoproteins in plants , especially for therapeutic purposes , requires modification of plant - specific sugar sidechains into a mammalian type of glycans . the invention provides recombinant bfsh and bfshr by infecting stably transformed tobaccoplants capable of forming mammalian type of glycans , with recombinant tobacco mosaic virus tmv containing the genes for bfsh or bfshr . construction of single chain ( sc ) bfsh into pks (+) bluescript vector , construction of sc - bfsh - tmv and sc - bfsh - his - tmv in order to circumvent the need of simultaneous expression of the two separate genes of bfsh - alpha and bfsh - beta subunits in plants , we decided to construct a bfsh fusion gene . by overlap pcr we fused the carboxyl end of the beta subunit to the amino end of the alpha subunit ( without a linker ). in addition , we constructed a second sc - bfsh version carrying a 6 × his tag at the c - terminus of the alpha subunit , which will allow us to purify the recombinant protein from the plant . both , sc - bfsh and sc - bfsh - his constructs were subcloned into the cloning vector pks (+) bluescript . the correctness of the clones was confirmed by sequence analysis . sc - bfsh was subcloned into the tmv vector . two positive clones were chosen to make in vitro transcripts and inoculate n . bentahamiana plants . after a few days , plants showed typical viral infection symptoms , which suggested the infective capacity of the recombnant tv clones . in order to test whether the sc - bfsh rna is stably expressed in systemically infected leaves , 8 days post inoculation rna was isolated from infected n . benthamiana leaves and a reverse transcriptase polymerase chain reactions using bfsh specific primers was performed . in all cases we obtained a pcr fragment of the expected size , indicating the stability of our sc - bfsh - tmv construct . extracts of infected plants are used for western blot analyses and elisa to determine whether sc - bfsh is expressed and folded properly . molecular cloning of full length cdna encoding the bovine fsh receptor ; cloning of the extracellular domain of the fsh receptor in tmv vector . oligonucleotide primers based on partial published sequence date were designed for pcr amplification of nucleotide 1 to 1100 and 650 to 2150 , respectively , from a bovine testicular cdna library . the two fragments were subcloned in the pgem - t vector ( stratagene ), and fully sequenced . a unique common internal restriction site ( xbal ) allowed the fragment ligation while subcloning into the eukaryotic expression vector pee14 . after plasmid amplification of a recombinant clone , transfection of cho - k1 cells is the next step . stable transfectants are usually obtained in three weeks . functional experiments ( hormone specific binding and transduction ) will allow selection of the best expressing clones before the amplification process with increasing amounts of msx in cell media . in order to obtain a soluble fsh receptor , a fragment encoding part of its n - terminal extracellular domain was obtained by using pcr . the size of the soluble receptor ( 293 aminoacids ) has been chosen in order to retain all hormone interaction , and favor processing by elimination of the c - terminal cystein cluster . amplimers bearing appropriate restriction sites for subcloning the tmv vector were designed . after amplification and cloning , a synthetic dna encoding the flag epitope ( sequence = dykddddk ) as well as a stop codon was ligated . subcloning of the construct into the tmv vector is now in progress . in order to express the bovine fsh in a transient plant expression system the following constructs were cloned in a tmv expression vector : a bfsh single chain ( sc - bfsh ) with the carboxy end of the β subunit fused to the amino end of the α subunits ( according to sugahara et al ., 1996 ) after inoculation of nicotiana benthamiana plants with in vitro transcripts from these constructs in all cases systemic infection of the recombinant viral constructs were obtained . by reverse transcription - pcr analysis we could demonstrate the in planta stability of the hybrid tmv genomes carrying the bfsh sequences . surprisingly also the co - transfected alpha and beta bfsh constructs were stably propagated in the same plants . for detection of recombinant bfsh western blot analysis of crude protein extracts from leaf material was carried out . using an anti - human fsh beta subunit antiserum ( fig5 ) we could demonstrate the expression of the β bfsh ( also in α / β cotransfected plants ), as well as the expression of the sc - bfsh and the sc - bfsh - his . the beta - bfsh appeared as a double band at about 14 kda . a major band at about 30 kda was observed for the sc - bfsh and sc - bfsh - his . no signals were observed in tmv - infected extracts . the presence of the 6 × his tag on the sc - bfsh could be demonstrated using anti - his monoclonal antibodies . in a small scale protein miniprep using ni - nta agarose we were able to purify the sc - bfsh - his under denaturing conditions . in order to investigate whether the sc - bfsh is glycosylated or not an enzymatic glycan digestion , pngasef digestion , was carried out . a clear band shift of bfsh treated with pngasef on western blot analysis indicated the presence of n - glycans . as sc - bfsh was detected almost exclusively in the soluble fraction after fractionating crude protein extracts with 100 . 000 × g , clearly the sc - bfsh is secreted by , the plant cells into the extracellular space . intercellular washing fluit ( if ) extractions from leaf material were carried out . as shown by western blot analysiss the sc - bfsh was clearly enriched in these if fractions indicating a secretion of protein into the extracellular space . expression of biologically active glycoforms of bovine follicle stimulating hormone in plants . the follicle - stimulating hormone ( fsh ) is a pituitary glycoprotein hormone which regulates the ovarian follicle and testicular tubule development in all vertebrate species . in particular ovine , porcine or equine fsh are widely used to induce superovulation for human assisted reproduction of cattle and can benefit from homologous ( i . e . bovine ) recombinant fsh being free of potentially infectious material and other contaminating hormones . here we describe the application of a plant based transient expression system for the rapid production of bfsh and its biochemical , immunological and biological . characterisation . we have used a tobacco mosaic virus - based vector to express bfsh in the tobacco related species nicotiana benthamiana . the genes encoding the beta and alpha subunits were introduced in tandem into the viral vector to produce a single - chain bfsh ( sc - bfsh ) protein . n . benthamiana plants infected with recombinant viral rna secreted high levels , up to 3 % of total soluble proteins , of sc - bfsh to the extracellular compartment ( ec ). in - situ indirect immunofluorescence revealed consistently that the plant cell is capable of efficiently targeting the mammalian secretory protein to the extracellular destination . mass spectrometry analysis of the n - glycans of immunoaffinity purified sc - bfsh derifed from ec fractions revealed two species of the plant paucimannosidic glycan type , derivatives of complex - type n - glycans . crude nonpurified protein extracts from the ec were used for in vitro and in vivo bioactivity assays . the sc - bfsh exhibited bioactivity as it was able to induce camp production in cho cell line expressing the porcine fsh receptor . furthermore , in superovulatory treatments of mice , sc - bfsh displayed significant in vivo bioactivity , although comparably low with respect to pregnant mare serum gonadotropins . we conclude that the rapid expression system used in this work may have a broad utility for the application of plant derived animal proteins in pharmaceutical products even for proteins where glycosylation is essential for function . the follicle - stimulating hormone ( fsh ) is a pituitary glycoprotein hormone which regulates the ovarian follicle and testicular tubule development in all vertebrate species ( pierce and parsons , 1981 ; bielinska and boime , 1995 ). together with luteinizing hormone ( lh ), thyroid stimulating hormone ( tsh ) and chorionic gonadotropin ( cg ), fsh forms the glycoprotein hormone family , which is the structurally most complex hormone family in the animal kingdom . these hormones are composed of two non covalently associated subunits , a common alpha subunit , and an unique beta subunit which confers biological specificity to each of these hormones . each subunit forms intrachain disulfide bridges and carries two n - linked oligosaccharides , which is necessary for proper folding and secretion of the hormones ( suganuma et al ., 1989 , feng et al ., 1995 ). the n - linked carbohydrate chains of fsh exhibit considerable variation in both size and structure , including the degree of terminal sialyation and / or sulfation ( baenzinger and green , 1988 ). the functional significance of this diversity of isoforms is not yet fully understood , but sialic acid seems to be the major determinant for the circulatory stability of fsh by preventing its rapid clearance mediated by the hepatic asialo - glycoprotein receptor ( drickamer , 1991 ). besides this influence upon plasma halflife , the glycan heterogeneity is thought to provide a fine tuning mechanism with which to control gonadal function . the technique of superovulation and embryo transfer is widely used to increase the number of offspring of genetically superior cows . induction of superovulation is usually performed using pregnant mare serum gonadotropin ( pmsg ) or pituitary derived fsh . apart from containing potentially infectious viral or prion material , these preparations have the disadvantage of always containing some lh which is thought to contribute to the high variation in the results of treatments ( wilson j m et al ., 1993 ). to overcome these problems , an attempt was made to produce recombinant bovine fsh ( rbfsh ) in the baculovirus expression system ( van de wiel et al ., 1998 ). two main problems were associated with rbfsh : first , the glycans apparently did not contain terminal sialic acid , due to a probable complete inability of the insect cell line to perform sialyation ( for a review see : altmann et al ., 1999 ). second , although a relatively high yield of rbfsh was obtained in the baculovirus expression system , sufficient upscaling of production has not been achieved yet , especially because a decrease of expression rates in large scale fermentations using higher cell densities ( taticek et al ., 1994 ). in the recent years , the expression of recombinant proteins in plants has become a matter of interest . as an eucaryotic system , plant cells are capable of targeting recombinant proteins to the secretory pathway and of carrying out posttranslational modifications including disulfide bridge formation and glycosylation ( hiatt et al . 1989 ; ma et al ., 1995 , cabanes - macheteau 1999 ). the n - glycosylation in higher organisms is conserved but differs in details . the processing of n - linked glycans occurs along the secretory pathway and complex - type n - glycans arise and are modified in the golgi apparatus . since some of the modifications are specific for an expression system , the structure of mature complex n - glycans differ to some extent in plants and mammals . in particular , plant glycoproteins do not bear sialic acid and carry a ( 1 , 3 )- fucose and b ( 1 , 2 )- xylose attached to their proximal n - acetylglucosamine which have not been found in mammals ( for recent review see lerouge et al ., 1998 ). since plants are gaining acceptance for the expression of recombinant therapeutic proteins ( e . g . ma et al ., 1998 tacket et al ., 2000 ), it is important to examine in detail to what extent glycans of mammalian glycoproteins produced in plants differ from the original ones , and could influence their physiological properties . in this instance , glycoprotein hormones offer a particularly demanding model since proper n - glycosylation is required for folding , subunit assembly , intracellular trafficing and biological activity . in this study , we used a plant viral vector to transiently express the bovine fsh in the tobacco related species nicotiana benthamiana . this viral system uses a hybrid tobacco mosaic virus ( tmv ) to express foreign genes systemically in whole plants ( casper and holt 1996 ). the levels of proteins expressed from tmv - based vectors are generally much higher than that obtained by stably transformed transgenic plants ( kusnadi et al ., 1997 , mccormick et al ., 1999 , krebitz et al ., 2000 ). another advantage of this system is the speed of recombinant protein production , which is based on the rapid systemic movement of tmv in plants . genes encoding the beta and alpha subunits were introduced in tandem into the viral vector to produce a single - chain bfsh ( sc - bfsh ) protein . using different approaches , such as biochemical fractionation and in - situ indirect immunofluorescence , we were able to show that the mammalian protein is targeted to the secretory pathway and is efficiently secreted to the periplasmic space of the n . benthamiana cells . using mass spectrometric a detailed n - glycan structure profile of the immunoaffinity - purified plant produced hormone was obtained . furthermore , using crude sc - bfsh extracts we demonstrated in vitro bioactivity , through receptor binding and activation of cho cells , and in vivo bioactivity , through superovulation of fecundable oocytes in mice . in order to construct the single chain bfsh ( sc - bfsh ) with the carboxyl end of the b - subunit fused to the amino end of the a subunit ( sugahara et al ., 1996 ) a gene soeing strategy ( horton , 1993 ) was chosen ( fig1 ): the bfsh a subunit was amplified from the plasmid bovalpha - psp64 # 1 ( leung et al ., 1987 ) using the primers fsh - f 5 ′- gga aat caa aga att tcc tga tgg aga gtt tac aat gca g - 3 ′, containing 13 bp of the b subunit &# 39 ; s carboxyl end , and nsi - stop 5 ′- agc tat gca tct att agg att tgt gat aat aac a - 3 ′. the bfsh b subunit was amplified from the plasmid bov fshbeta pgem3 ( maurer and beck , 1986 ) using the primers fsh - a 5 ′- ata tga gtc gac atg aag tct gtc cag ttc - 3 ′ and fsh - e 5 ′- ctc cat cag gaa att ctt tga ttt ccc tga agg agc agt a - 3 ′, the latter including 13 bp of the a 5 ′- end . the resulting 2 fragments which contain a 26 bp overlapping region were combined in 5 pcr extension cycles with an annealing temperature of 45 ° c . subsequently , this overlap pcr product was amplified by pcr using the primers fsh - a and nsi - stop . following sali / nsii digestion , this fragment was ligated to sali / psti restricted tmv based expression vector p4gd - pl ( casper and holt , 1996 ), resulting in the construct p4gd - sc - bfsh . this construct was used for all expression experiments . [ 0085 ] nicotiana benthamiana plants were grown in a controlled growth chamber with 22 ° c . day and night temperature , 50 % humidity and 16 h light period . the recombinant viral vectors p4gd - sc - bfsh and p4gd - pl ( as negative control ) were linearized by sfii digestion . capped in vitro run off transcripts were made using a t7 transcription kit ( ribomax , promega ltd ., wi , usa ). in vitro transcripts were used to mechanically inoculate n . benthamiana plants at a six leaf stage . symptoms of infections were visible 8 - 10 days post inoculation ( dpi ) as leaf deformations , with some variable leaf mottling and growth retardation . 12 - 16 dpi the replicative stability of the hybrid tmv rna — genome derived from p4gd - sc - bfsh was investigated . total rna from systemically infected leaves was prepared using trireagent ( molecular research centre , inc .) and cdna synthesis ( reverse transcription ) was performed using the tmv ( p4gd - pl ) specific reverse primer p4gd - rv 5 ′- ttt ttc cct ttt ttg ttt tcc g - 3 ′ located downstream the multiple cloning site . using p4gd - rv and the tmv specific forward primer p4gd - fw 5 ′- gat gat gat tcg gag gct act - 3 ′ which anneals upstream of the multiple cloning site , a specific rt - pcr fragment of 826 bp was expected for the sc - bfsh construct . as negative control the same procedure was also carried out on wt tmv ( p4gd - pl ) infected plants . in this case a rt - pcr product of 145 bp was expected . total soluble protein extraction from sc - bfsh expressing n . benthamiana leaves two to three weeks after inoculation , systemically infected leaves were harvested , and total soluble protein ( to ) was extracted by grinding the leaves in 10 vols ( w / v ) of 10 mm tris - hcl ph 7 . 6 buffer . cellular debris were sedimented ( 15 min , 6000 rpm , 4 ° c .). the supernatant was used for sds - page . the same procedure was also carried out on leaves which were subjected to intercellular washing fluid extraction ( see below ). in order to enrich periplasmic ( extracellular ) proteins an intercellular washing fluid ( if ) extraction was carried out using the method described by pogue et al . ( 1997 ) with modifications . systemically infected leaves from sc - bfsh expressing plants , were harvested two to three weeks after inoculation . the tissue was rolled lengthwise in parafilm ® ( american national can , il , usa ), inserted into a 50 ml plastic tube and submerged in pre - cooled ( 4 ° c .) 10 mm tris - hcl ph 7 . 6 buffer . a vacuum was applied twice for 1 min , with a rapid release of the vacuum to infiltrate the leaves with buffer . subsequently , the leaves were blotted dry to remove excess buffer , were again rolled in parafilm and inserted into a centrifuge tube . the if was collected by a downspin at 3 , 000 × g for 15 min at 4 ° c . the if was finally clarified by a centrifugation at 10 . 000 × g for 15 min at 4 ° c . and the supernatant used for analyses . the same if preparations were made from plants infected with p4gd - pl ( wt tmv ) or not infected wildtype plants serving as negative controls . crude total protein extracts ( to ) from sc - bfsh expressing plants were prepared as described above , followed by ultracentrifugation at 100 . 000 × g for 1 h at 4 ° c . to obtain a pellet ( p ) and a supernatant ( sn ) fraction . the pellet was resuspended in 10 mm tris - hcl ph 7 . 6 in the same volume as the supernatant fraction . equal volumes of total extract , pellet and supernatant fractions were subjected to western blot analysis ( see below ). the procedure was as described by tretter et al ., 1991 with modifications . if extraction from sc - bfsh expressing plants ( see above ) was conducted using 50 mm tris - hcl , 20 mm edta , ph 8 . 0 , followed by an addition of b - mercaptoethanol and sds to each 0 , 5 % ( v / v , w / v , respectively ). 10 ml of this extract were heated at 95 ° c . for 5 min , followed by an addition of 40 ml 50 mm tris - hcl , 20 mm edta , ph 8 . 0 and 1 % igepal ( sigma , mi , usa ). finally , 1 ml of n - glycosidase f , peptide - n4 -[ n - acetyl - b - glucosaminyl ] asparagine amidase , ( 20 mu / ml ; roche , ch ) was added , followed by an incubation at 37 ° c . overnight ( 16 h ). after digestion , the proteins were precipitated using aceton and subjected to western blot analysis . as a control , the same procedure was done without the addition of n - glycosidase f . protein extracts prepared as described above were electrophoresed on 12 . 5 % sds - polyacrylamide gels under reducing conditions ( lämmli , 1970 ). following electroblotting onto nitrocellulose ( amersham life science ltd , u . k . ), the blots were blocked with 5 % non - fat dry milk in tbs containing 0 , 1 % tween 20 ( sigma , mi , usa ). the primary antibody was an anti - human fsh b - subunit ( r812 , reference !) rabbit polyclonal antiserum diluted to 1 : 2500 in tbs containing 0 , 1 % tween ( ttbs ) and 1 % bsa ( sigma , mi , usa ). as secondary antibody an anti - rabbit igg goat polyclonal antiserum - horde radish peroxidase conjugate ( sigma , mi , usa ), diluted to 1 : 20000 in ttbs , was employed . detection was done using an enhanced chemiluminescence substrate ( ecl , super signal , pierce , ill ., usa ). for quantification of the signal intensities kodak digital science 1d image analysis software was used . in parallel to the immunodetections , the total protein contents of the different extracts were visualised using silver staining of the gels ( amersham pharmacia biotech ab ). quantitation of total protein was done using a bca protein assay kit ( pierce , ill ., usa ). indirect in situ immunofluorescence was performed according to goodbody et al . ( 1994 ) and flanders et al . ( 1990 ) with modifications . all solutions were made in microtubule stabilizing buffer ( mtsb ): 50 mm pipes , 5 mm egta , and 5 mm mgso 4 , ph 6 . 9 . epidermal tissue sections ( leaf stalk ) from sc - bfsh expressing plants , prepared as described above , were fixed in 4 % ( v / v ) formaldehyde for 60 min , followed by four washing steps in mtsb over 60 min . in order to aid antibody penetration , the tissue was cross hatched with a flexible , double sided razor blade . permeabilisation was performed by 10 % dmso and 0 . 4 % igepal ( sigma ) for 15 min . an additional blocking step was performed by incubating in 1 % bsa for 15 min . both antibody incubations were carried out for 60 min followed by three washing steps with the blocking solutions after the primary incubation and with mtsb after the secondary incubation . as primary antibody the rabbit anti - hfsh — b — peptide polyclonal antibody ( r812 ) was used , the secondary antibody was a cy3 conjugated sheep anti - rabbit antibody ( sigma ). as anti - fade mounting reagent , citifluor was used ( city university , london ). imaging was conducted on a biorad mrc 600 confocal laser scanning microscope equipped with a krypton / argon mixed gas laser and a × 40 objective . excitation of the cy3 fluorochrome was done at 569 nm using the yhs filter block . phase contrast illumination of the tissue sections was performed on the same sections . images from the confocal system were imported into paintshoppro 5 . 01 ( jasc software , inc ., mn , usa ) for colorisation . immunoaffinity chromatography was essentially done as described previously ( van de wiel et al ., 1998 ) and according to the mannufacturer &# 39 ; s instructions . the gel pellet was washed 2 times with pbs13 and then incubated with 30 ml of sc - bfsh the purity and concentration of sc - bfsh in the eluate fractions were monitored on a silver stained sds - page - gel , on which the sc - bfsh appeared as a single band . for mass spectrometry analysis ( see below ), a volume of pure sc - bfsh corresponding to approximately 8 mg was first dialyzed against deionized water using slide - a - lyzer ® mini dialysis units having a molecular weight cut off of 10 kda ( pierce , ill ., usa ) to remove salts and glycine . subsequently , the dialyzed fractions were concentrated by lyophilisation , and dissolved in sds - page sample buffer . n - glycan analysis of sc - bfsh by matrix assisted laser desorption / ionisation mass spectrometry ( maldi - ms ) the procedure was carried out as described by kolarich and altmann ( in press ) with modifications . in brief , approximately 8 mg of immunoaffinity — purified sc - bfsh were electrophoresed on a reducing 12 . 5 % c 1 % t sds - polyacrylamide gel system . following electrophoresis , the gel was stained using a silver staining method which is compatible with mass spectrometry analysis ( schevchenko et al . 1996 ). the band of interest was excised with a scalpel , and after washing , reduction and s - carboxamidomethylation subjected to tryptic in - gel digestion as described by jensen et al . ( 1997 ). in order to identify the sc - bfsh , the extracted peptides were dissolved in 10 ml 5 % ( v / v ) formic acid and analysed on a dynamo ( thermobioanalysis , ltd .) linear time - of - flight maldi - ms ( peptide mapping ). 0 . 2 ml of the sample was dried on the plates followed by addition of 0 . 8 ml matrix solution ( 1 % ( w / v ) a - cyano - 4 - hydroxycinnamic acid in 70 % ( v / v ) acetonitrile . peptides were measured with a & lt ;& lt ; dynamic extraction & gt ;& gt ; setting 0 . 1 . average masses of [ m + h ] + ions were determined using human bradykinin and human renin substrate tetradecapeptide for external calibration of the instrument . the expasy & lt ;& lt ; peptide - mass & gt ;& gt ; program was used to construct theoretical peptide maps of sc - bfsh . following peptide mapping , the n - glycans in the residual aliquot of the tryptic digest were released by peptide - n 4 -( n - acetyl - b - glucosaminyl ) asparagine amidase a ( n - glycosidase a , roche , ch ). to remove peptides and salts , the digest was loaded onto a triphasic microcolumn consisting of anion exchange , reversed phase and a mixture of polyamide / cation exchange resins . for maldi - ms analysis , the samples were redissolved in 10 ml water . 1 ml of the sample was spotted onto the target , dried under vacuum , followed by addition of 0 . 8 ml matrix solution ( a 1 : 1 : 1 mixture of 2 % ( w / v ) 2 , 5 - dihydroxybenzoic acid in 30 % ( v / v ) acetonitrile , 1 % ( v / v ) d - arabinosazone in acetonitrile and 0 . 2 m 2 , 5 - dihydroxybenzoic acid / 0 . 06 m 1 - hydroxy - isoquinoline in 50 % ( v / v ) acetonitrile ( darci ). the oligosaccharides were analyzed with a & lt ;& lt ; dynamic extraction & gt ;& gt ; of 0 . 1 . compilation of the spectra was done manually by the addition of single shots . average masses of [ m + na ] + ions were recorded using a partial dextran hydrolysate for external calibration of the instrument . if - extracts from n . benthamiana plants infected by p4gd - sc - bfsh or p4gd - pl ( negative control ) were diluted in gmem - s medium without calf serum in a final volume of 0 . 2 ml as indicated in fig8 a . these extract dilutions were incubated on cho cell layers expressing the porcine fsh receptor ( abdennebi et al ., 1999 ) for 1 h 30 at 37 ° c . known concentrations of pituitary bfsh were applied to cells in the same conditions ( see fig8 b ). camp levels in supernatants were determined using a specific ria ( nen - dupont de nemours , les ulis , france ). all assays were performed in duplicate and repeated twice . 15 6 - 8 week old female c57 / cba mice were treated each with 100 ml of 11 times concentrated sc - bfsh - if extract ( concentration was done using an amicon ultrafiltration cell , mwco 10 kda ). serving as a negative control , 15 mice were treated each with 100 ml of 11 times concentrated if - extract from not infected plants ( ni - if ). furthermore , the response of 14 mice to 5iu pregnant mare serum gonadotropin ( pmsg , folligon , intervet ) in the same volume was investigated . 46 - 48 hours post fsh or wt - if injections , the 3 groups were treated with 5 iu human chorionic gonadotropin ( hcg ; chorulon , intervet ). to recover mature oocytes , superovulated females were sacrified 15 hours post - hcg - injection . oocytes were incubated after collection in 0 . 5 % hyaluronidase ( sigma ) in pbs for 1 - 2 min at 37 ° c . to remove cumulus . the total number of oocytes were counted . vector construction and expression of sc - bfsh in n . benthamiana plants although native bfsh is expressed from two different genes on different loci , we chose to genetically fuse the carboxyl end of the bfsh b subunit to the amino end of the a subunit according to sugahara et al . ( 1996 ) in order to produce a single - chain bfsh ( sc - bfsh ). both the receptor binding affinity and the potency of adenylate cyclase activation for the single - chain human fsh were shown to be similar to that of recombinant human fsh heterodimer ( sugahara et al . 1996 ). the fusion gene was inserted into the tobacco mosaic viral - based vector p4gd - pl ( casper and holt 1996 ). in vitro run off transcripts of the construct p4gd - sc - bfsh were capable of infecting n . benthamiana plants systemically as indicated by clear mottling and mosaic symptoms on systemic infected leaves 10 - 14 days post inoculation ( dpi ). to determine the inplanta replicative stability of the hybrid tmv rna , reverse transcription pcr ( rt - pcr ) was carried out with rna isolated from newly developed leaves 14 dpi . the amplification of a single rt - pcr fragment of expected size confirmed the presence of the fsh sequence in systemically infected tmv leaves . further rt - pcr analyses were carried out until 28 dpi , in which likewise no instabilities of the p4gd - sc - bfsh derived viral rna were observed . 17 - 21 dpi infected leaves were harvested , total soluble protein ( to ) extracted and subsequently subjected to sds - page . silver staining revealed the abundant coat protein at position 17 kda and a diffuse additional band at position 30 kda , the expected size of undissociated alpha - beta bfsh heterodimer ( wu et al ., 1992 ), which was not present in extracts of control plants . the corresponding western blot analysis using an anti - humanfsh beta ( hfshβ ) antiserum clearly confirmed the expression of the hormone by displaying a strong signal at position 30 kda . minor signals were obtained at 60 kda which we interpret as artefactual dimerisation product that can occur during sds - page ( shi and jackowski , 1998 ). additionally , a putative degradation product of sc - bfsh was detected at approximately 15 kda . it was not possible to reduce the amount of this degradation product by including a cocktail of plant protease inhibitors to the extraction buffer . the specificity of the signals obtained in sc - bfsh expressing plants was indicated by the absence of any signal in extracts from control plants . in a series of experiments using different approaches we wanted to investigate whether the sc - bfsh as a glycoprotein hormone — in homology to the situation in mammals — is targeted to the secretory pathway of plant cells and if the protein is secreted into the periplasmic ( extracellular ) space . ultracentrifugation of total protein extracts ( to ) from sc - bfsh expressing plants was carried out which resulted in a pellet ( p ) and a supernatant ( sn ) fraction . all three fractions , to , p and sn , were subsequently analysed by sds - page . the different staining patterns of sn and p on a silver - stained gel indicated a selective enrichment of soluble and of mostly membrane associated proteins , respectively . silver staining and immunoblot analyses using anti - hfshb antibodies clearly revealed the enrichment of the hormone in the soluble sn fraction , which is consistent with a periplasmic location . the fact that no hormone - was detected in the p fraction excludes the formation of inclusion bodies which often is a consequence of protein overexpression . as a next step a so - called intercellular washing fluid ( if ), which is characterised by the specific enrichment of periplasmic ( extracellular ) proteins , was separated from the total protein extracts ( to ) and compared with the remainder ( re ) thereof . to , if and re fractions were subjected to sds - page and silver staining revealed an additional diffuse band in the if at the expected size of the hormone ( 30 kda ), which is absent in control if fraction ( fig3 ). immunoblotting clearly demonstrated the enrichment of the sc - bfsh in the if fraction . we calculated an enrichment factor of 6 - 10 for sc - bfsh in the if with respect to the to fraction . furthermore , the computer - assisted comparison of the signal intensities of 50 ng pit - bfsh with that of sc - bfsh in to and if fractions allowed us to estimate sc - bfsh concentrations of 0 . 4 % and 3 % of total soluble protein , respectively . to confirm the periplasmic location of sc - bfsh , in situ indirect immunofluorescence was performed . mechanical sectioning of epidermal cells of sc - bfsh expressing plants was used to provide entry sites for the antibodies . hence , only cut cells show an immunostaining . the fluorescence signal was obtained in the periphery of the cells , being clearly different from a cytoplasmic or vesicular fluorescence staining as shown previously ( boevink et al ., 1998 ; essl et al ., 1999 ). the in situ indirect immunostaining of sc - bfsh appeared as a thin film located inside of the cell wall being consistent with a periplasmic location . since native bfsh is a glycprotein hormone and its n - glycosylation is essential for bioactivity , the n - glycosylation status of sc - bfsh was investigated . sc - bfsh has four potential n - glycosylation sites and , in western blot analyses , a diffuse band was detected at position 30 kda , which is larger than the expected size of the unglycosylated ( 23 kda ) protein ( fig2 ). this already indicated the glycosylated status of the recombinant protein . as a first approach a glycan specific enzyme ( pngase f ) digestion of if extracts was made . pngase f digests all oligosaccharide species except those containing the plant specific core α1 , 3 fucose ( tretter et al ., 1991 ). clearly the band detected at position 30 kda shifted to a band of smaller size ( 26 kda ) indicating sensitivity of sc - bfsh to n - glycosidase f . this result demonstrated the presence of n - linked glycans lacking core α1 , 3 fucose . in addition , presence of a minor “ smear ” signal at position 26 - 27 kda which is not susceptible to n - glycosidase f digestion , indicates the presence of a fraction carrying core α1 , 3 fucose residues . the detailed structure of n - glycans attached to sc - bfsh was elucidated by maldi - ms . this procedure was specially designed for the analysis of n - glycans potentially containing core fucose in a1 , 3 linkage . immunoaffinity — chromatography using a monoclonal antibody against human fsh was carried out to purify the sc - bfsh from if extracts , resulting in & lt ;& lt ; single band purity & gt ;& gt ; as evidenced by sds - page / silver staining . the sc - bfsh was subjected to tryptic in - gel digestion in order to provide susceptibility to the subsequent n - glycosidase a digestion . this further allowed the identification of the tryptic peptides measured by maldi - ms ( data not shown ). subsequently , the enzymatically released n - glycans were cleaned up for maldi - ms . the resulting mass revealed two peak masses that could be assigned to 2 known plant n - glycans of the paucimannosidic type : mmx and mmxf 3 . an analysis of the respective peak areas revealed a 4 : 1 ratio between mmx and mmxf 3 . consistent with the result of the n - glycosidase f digestion , the mass spectrometric analysis revealed a glycan species , mmx , which is susceptible to n - glycosidase f digestion , and a second minor fraction , mmxf 3 , which is not . to determine the in vitro bioactivity , the plant - expressed sc - bfsh was tested for its ability to induce cyclic amp ( camp ) production in a cho cell line expressing the porcine fsh receptor ( pfshr ). evidently , camp levels , as determined by ria , were raised in a dose dependent manner upon addition of increasing amounts of pit - fsh . the effect of the plant expressed sc - bfsh on the production of camp in this cell line was determined by applying several nonpurified sc - bfsh - if dilutions . increasing concentrations of the sc - bfsh containing if extract resulted in a dose responding camp production . the specificity of the camp production upon addition of plant - produced sc - bfsh was demonstrated by an absence of a response of the cells to an if extract from control plants . the pit - bfsh standard curve allowed to estimate a concentration of 5 ng in vitro bioactive sc - bfsh per ml if . 15 6 - 8 week old female c57 / cba mice were treated each with 100 ml of 11 times concentrated sc - bfsh - if extract ( concentration was done using an amicon ultrafiltration cell , mwco 10 kda ). serving as a negative control , 15 mice were treated each with 100 ml of 11 times concentrated if - extract from not infected plants ( ni - if ). furthermore , the response of 14 mice to 5 iu pregnant mare serum gonadotropin ( pmsg , folligon , intervet ) in the same volume was investigated . 46 - 48 hours post fsh or wt - if injections , the 3 groups were treated with 5 iu human chorionic gonadotropin ( hcg ; chorulon , intervet ). to recover mature oocytes , superovulated females were sacrified 15 hours post - hcg - injection . oocytes were incubated after collection in 0 . 5 % hyaluronidase ( sigma ) in pbs for 1 - 2 min at 37 ° c . to remove cumulus . the total number of oocytes were counted . the total number of oocytes indicated a high superovulatory response of the mice to pmsg , where as much as approximately 4 fold more oocytes were counted as compared to the negative control . a significant , albeit comparably low , superovulatory response to sc - bfsh ( 1 , 5 fold above the negative control ) was found . the mean number of oocytes for each group , wt - if , sc - bfsh - if and pmsg , and the respective standard deviations are illustrated . female mice were treated with intraperitoneal injection of pregnant mare serum gonadotropin ( folligon - intervet , 5 iu ), sc - bfsh - if extracts , wt - if extracts , followed by human chorionic gonadotropin ( chorulan , intervet ) 48 h later . as a control , untreated females that showed an oestrus behaviour were included in this study . mice were caged overnight with males and 1 cell stage embryos were isolated 19 - 26 hours after hcg from females showing sperm vaginal plugs ( day 1 ). the ampullary regions of excised oviducts was placed at 30 ° c . in pbs medium containing bovine serum albumine at 4 mg / ml together with bovine hyaluronidase ( sigma ) at 50 units / ml . after 3 - 5 minutes the cumulus cells were dissociated and the eggs washed several times in pbs medium . fertilized eggs showing two pronuclei and polar body were pooled from several females of the same group . embryos were cultured under paraffin oil ( dbh ) in 10 μl drops of whitten &# 39 ; s medium in an atmosphere of 5 % co2 in air at 37 ° c . embryos were cultured for up to 72 hours in vitro in whitten &# 39 ; s medium and examined several times . for each group , development was assessed by the proportion of fused eggs that became blastocysts . the results clearly indicated that , whatever the treatment , more than 80 % of the fused eggs reached the morula stage 3 , 5 days after fecondation while at 7 , 5 days more than 50 % of them developed into bastocysts . our results clearly showed no differences between pmsg and plant fsh extract treated females . these experiments strongly suggested that crude extract of infected plants containing fsh did not induce any deleterious effects on mice embryo further development , indicating potential use for assisted reproduction . two different assays were performed to detect the presence of sc - bfsh - fsh receptor complexe ( fshc ) in tobacco leaves . the wells of m96 microtiterplate were coated with polyclonal antiserum against fsh receptor ( dilution at 1 / 200 ). to each coated well , 100 μl was added of serial dilutions of either wt - if extract or intracellular fluid from infected tobacco leaves containing fshc . after incubation ( 1 h at 37 ° c .) and washing , a monoclonal antibody against human fshβ ( 0 . 01 mg / ml ) was added and incubated ( 1 h at 37 ° c .) which was followed by washing and addition of anti - mouse igg coupled to peroxidase ( 1 : 500 , 100 μl / well ) after washing , tmb and h 2 o 2 were added ( for color development ). the reaction was stopped by adding h 2 so 4 . the assay used coated beads for the capture of fshc from infected tobacco leaves . polystyrene beads were incubated overnight at 4 ° c . in the presence of polyclonal antibody against fsh receptor . after washing with distillated water , beads were used to capture antibody - reactive molecules present in the if extract ( wt or fshc ). after 2 h incubation at followed by extensive washing , labeled diluted monoclonal antibody against fshp ( in 0 . 01 m pinacl containing 50 % foetal calf serum ) was added . the reaction mixture was incubated for 1 h at 20 ° c ., followed by a washing step and residual radioactivity was counted . here we demonstrated the rapid and high level expression of a single - chain version of the bovine follicle stimulating hormone ( sc - bfsh ) in nicotiana benthamiana plants using a tobacco mosaic virus based transient expression system . a combination of molecular and cell biological experimental approaches showed consistently that the plant cell is fully capable of directing a mammalian secretory protein such as a glycoprotein hormone to the extracellular destination . hence , the native leader sequence of the beta subunit of bfsh ( and accordingly also the btsh subunit ) which represents the n - terminus of the sc - bfsh is recognised by the plant cell and subsequently the protein is directed to the secretory pathway . this observation is in agreement with the correct recognition of mammalian signal peptides derived from antibodies by the plant cell machinery . furthermore , correct formation of disulfide bridges and folding of the tethered hormone subunits similarly to its native counterpart pituitary bfsh was evidenced by the in vitro bioactivity assay . most clinically important mammalian proteins , such as tsh , fsh and lh , have n - glycans , which confer different biological functions , such as resistance to protease attacks , antigenicity , immunogenicity and , as for fsh , plasma clearance rates . although n - glycosylation is conserved in higher organisms to some extent , so far no established heterologous expression system produces correct mammalian - type n - glycans , due to more or less differing biosynthetic pathways . the perspective to use plants as economic factories to produce therapeutic recombinant proteins at a low cost makes it important to investigate the capacity of plant cells to produce functional mammalian - like glycoproteins . our detailed analysis on the n - glycosylation pattern of sc - bfsh constitutes a complete study of a mammalian glycoprotein . surprisingly , only two oligosaccharide structures were found n - linked in sc - bfsh and were identified as paucimannosidic n - glycans containing b1 , 2 xylose and a1 , 3 fucose residues in a ratio 80 : 20 %, respectively . paucimannosidic glycans are considered as typical vacuole - type n - glycans , which result from the elimination of the terminal residues of complex - type n - glycans in post - golgi compartments ( for review see lerouge et al . 1998 ). secreted proteins in plants usually carry complex - type n - glycans with a high degree of heterogeneity ( melo et al ., 1997 , ogawa 1996 , for a review see sturm 1995 ). although paucimannosidic n - glycans have been found to a minor extent in secreted proteins , the presence of this type of glycan as predominant species is a rather unusual case . still , to our knowledge , there is only one detailed comparative study of a mammalian glycoprotein , a mouse immunglobulin (“ plantibody ”), produced in a plant expression system ( cabanes - macheteau et al ., 1999 ). in contrast to sc - bfsh the plantibody shows a higher degree of n - glycan heterogeneity , as a total of 8 different species of oligosaccharides were found . however , since no detailed analysis of the protein location was done it cannot be excluded that fractions of plantibodies are stored in different compartments of the plant cell ( cabanes - macheteau et al ., 1999 ). to our knowledge this is the first report of a detailed n - glycan analysis of a protein derived from an if fraction , usually secreted proteins are analysed from total protein fractions . this might be a reason why less heterogeneity was found . evidently , the n - glycans present on sc - bfsh exhibit considerable structural aberration from its native counterpart , pituitary bfsh ( baenzinger and green , 1988 ). as anticipated from known plant n - glycan structures , no n - glycans of the mammalian complex - type were found , neither b1 , 4 linked galactose nor terminal sialic acid . b ( 1 , 2 ) xylose and core a ( 1 , 3 ) fucose have never been found in mammals cells and they are considered potentially immunogenic structures ( wilson et al ., 1998 ; kurosoka et al ., 1991 ; faye et al ., 1993 ??). although so far no negative effect has been reported for plantibodies applied to mammals which might result from these sugars , no long term studies are available . we showed evidence , that the plant - produced hormone has in vivo bioactivity . as appointed above , another important aspect of these experiment is the fact that the application of a highly concentrated if extract , which comprises a complex mixture of periplasmic proteins , did not have an deleterious effect on the model animal . unlike other established protein expression systems , such as bacterial , yeast or animal cell culture systems , plant if extracts may be directly applicable in acute medical treatments without the need of further expensive purification . in summary we conclude that the tmv - based expression system provided here gives a very attractive expression system for mammalian glycoproteins such as glycoprotein hormones , since bioactive glycoforms of sc - bfsh accumulate to high levels in the periplasmic space of n . benthamiana leaves . we also could demonstrate the important benefit of being able to apply crude protein if - extracts without the concerns of an exposure to potentially infectious agents and apparently without any acute deleterious effect to the model animal . glcnac , n - acetylglucosamine ; fuc , fucose ; gal , galactose ; galt , beta 1 , 4 - galactosyltransferase ; rca , ricinus cummunis agglutinin ; [ 0150 ] fig1 major differences between mammalian and plant complex n - linked glycans . drawn are typical n - linked glycans . numerous variations , both extended or truncated , occur in mammals and plants . [ 0151 ] fig2 comparison of rna levels and product of β1 , 4 - galactosyltransferase . upper panel : northern blot of total rna isolated from 25 transgenic plants , including a not transformed control plant ( 0 ), detected with a human β1 , 4 - galactosyltransferase probe . lower panel : western blot of the same plant probed with rca to detect terminal galactose residues on glycoproteins . m . indicates the molecular weight marker . [ 0152 ] fig3 western blot showing the binding of lectin and antibody to protein isolated from wild - type and a β1 , 4 - galactosyltransferase plant ( no . 8 from fig2 ). a : rca as in fig2 b : anti hrp ( detecting both xylose and fucose ) antibody , c : anti xylose antibody , d : anti fucose antibody .) [ 0153 ] fig4 western blot showing rca and sheep - anti - mouse - igg binding to purified antibody produced in hybridoma culture ( hyb ), tobacco plants ( plant ) and tobacco plants co - expressing β1 , 4 - galactosyltransferase ( galt11 and galt12 ). h . c . : heavy chain , l . c . light chain . [ 0154 ] fig5 western blot showing specific antibody binding to recombinant beta - fsh and recombinant alpha - and beta - fsh expressed in plants . using an anti - human fsh beta subunit antiserum we could demonstrate the expression of the beta bfsh also in alpha / beta cotransfected plants . the beta - bfsh appeared as a double band at about 14 kda . [ 0155 ] fig6 in vivo bioassay for the determination of the activity of biopharmaceutical plant - derived glycoprotein hormone preparations in mice . superovulatory treatment of c57 / cba mice with sc - bfsh : the responses , i . e . numbers of counted oocytes , of 15 or 14 mice treated each with either sc - bfsh if extract corresponding to approx . 4 , 8 iu , or with equal amounts of if extract of not infected wildtype plants ( wt - if , negative control ) or with pmsg corresponding to each 5 iu of fsh ( positive control ) are listed in the table . the total ( sum ) and mean numbers of oocytes including the standard deviations for the three groups are given . the diagram illustrates the mean numbers of oocytes counted for the three groups of mice treated with sc - bfsh - if , wt - if or pmsg . the standard deviations are indicated ( sd ). a , b : aoki , d ., lee , n ., yamaguchi , n ., dubois , c ., and fukuda , m . n . 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