Patent Application: US-49920300-A

Abstract:
nucleic acid molecules encoding an alternansucrase are provided . moreover , vectors , host cells and plant cells transformed by the herein - described nucleic acid molecules and plants containing them are provided . furthermore , methods are described for preparing transgenic plants which synthesize the carbohydrate alternan , because of the insertion of nucleic acid molecules encoding an alternansucrase . moreover , methods for preparing alternan and products resulting from them are provided .

Description:
by the use of standard methods ( sambrook et al ., molecular cloning : a laboratory manual , 2nd issue ; cold spring harbor laboratory press , n . y ., usa ( 1989 )) we introduced a different polylinker ( see fig1 ) between the 35s promoter and the ocs - terminator into the plasmid pbinar ( höfgen und willmitzer , plant science 66 ( 1990 ), 221 - 230 ). the resulting plasmid was called pbinar - n . via standard methods ( sambrook et al ., molecular cloning : a laboratory manual , 2nd issue ; cold spring harbor laboratory press , n . y ., usa ( 1989 )) we isolated an ecori / hindiii - fragment from pbinar - n containing the 35s promoter , the polylinker and the ocs - terminator . this fragment was then ligated into the same restriction sites of plasmid pbib - hyg ( becker , nucleic acids research 18 ( 1990 ), 203 ). the resulting plasmid was called pbinar - hyg - n . by using the oligonucleotides sp - pat - 5 ′ and sp - pat - 3 ′ ( s . seq id nos . 48 and seq id no . 49 ) we amplified dna molecules coding for the leader peptide of the patatin protein from potato ( see seq id no . 50 , which differs from the sequence used by sonnewald et al . plant j . 1 ( 1991 ), 95 - 106 ) via a pcr approach using plasmid pgt5 ( rosahl et al ., mol . gen . genet . 203 ( 1986 ), 214 - 220 ; sonnewald et al ., plant j . 1 ( 1991 ), 95 - 106 ) as a template . the resulting pcr products were cut by the restriction enzymes xbal and sall and then ligated into the plasmid pbinar - hyg - n which was linearized before by using the restriction enzymes spel and sall . the resulting plasmid was called pbinar - pat - hyg . the steps 2 to 4 were repeated 35 times in a cyclical manner . by using the oligonucleotides sp - fnr - 5 ′ and sp - fnr - 3 ( see seq id no . 51 and 52 ) we amplified dna molecules coding for the transit peptide of the fnr protein from spinach via a pcr approach using plasmid p6socfnr - 15 ( jansen et al ., current genetics 13 , ( 1988 ), 517 - 522 ) as a template . the resulting pcr products were cut by xbal and sall and then cloned into the spel / sall - opened pbinar - hyg - n . resulting plasmid was called pbinar - fnr - hyg . buffer and polymerase from gibco brl ( platinum taq dna polymerase high fidelity no . 1304 - 011 ) the steps 2 to 4 were repeated 35 times in a cyclical manner . the strain leuconostoc mesenteroides nrrl - b1355 was cultured in 1 l of lactobacilli mrs broth ( difco ) complemented with 5 % saccharose at 28 ° c . for two days . after the culture was subjected to centrifugation at 20 , 000 × g for 30 minutes , the supernatant was admixed with the same volume of 10 % trichloro acetic acid and stirred at 4 ° c . for 16 hours . this solution was then subjected to centrifugation at 10 , 000 × g for 30 minutes . the thus obtained precipitate was dissolved in 4 . 5 ml of 40 mm tris - hcl , ph 8 . 8 , and subsequently neutralized with ( about 0 . 5 ml ) 2 m tris - base . this protein solution was given to the company toplab gesellschaft fur angewandte biotechnologie mbh , martinsried , germany , for protein sequencing . at this company , the protein solution was electrophoretically separated in sds polyacrylamide gel , the gel was stained with coomassie blue and the staining was subsequently removed by 10 % acetic acid . for the enzymatic digestion of the protein , the protein bands were cut from the gel , pressed through a sieve and fragmented ( pores 30 μm × 100 μm ). the crushed gel was then washed with half concentrated incubation buffer ( 12 . 5 mm tris , 0 . 5 mm edta ph 8 . 5 ) for 2 minutes . subsequently , it was subjected to centrifugation , the buffer was removed and the gel was dried in the “ speedvac ” for one hour ( about 5 % residual water , rubber - like ). subsequently , a solution of endoproteinase lysc in 400 μl 12 . 5 mm tris / hcl , ph 8 . 5 ( enzyme : protein = 1 : 10 ) and 0 . 1 % of laurylmaltosite was prepared . 200 μl of this solution were added to the sample and incubated in the heat block shaker at 37 ° c . overnight . in order to elute the peptide fragments , a one hour incubation with 1 % tfa was carried out , twice , followed by centrifugation , and subsequently by elution with 10 % formic acid , 20 % isopropanol , 60 % acetonitrile for 3 hours . the peptide fragments obtained were then separated from each other by hplc ( column superspher 60 rp select b ( merck , darmstadt ) 2 mm × 125 mm ; buffer a 0 . 1 % trifluoro acetic acid , buffer b : 0 . 085 % tfa in acetonitrile ; flow rate : 0 . 2 ml / min ; gradient : 5 - 60 % in 60 min ; detection at 206 nm . the peptide fragments obtained were then sequenced in an automatic sequencer procise 492 ( applied biosystems , pe ); the procedure being the stepwise edman degradation in a modification according to hunkapiller ( hunkapiller et al ., meth . enzymol . 91 ( 1983 ), 399 - 413 ). six different peptide sequences ( see seq . id nos . 5 to 9 , seq . id no . 21 ) were identified which were designated lysc - 66 , iysc - 67 , lysc - 82 , iysc - 83 , lysc - 88 and “ n - terminus ”. preparation of a genomic dna library from leuconostoc mesenteroides nrrl b1355 leuconostoc mesenteroides nrrl - b1355 ( purchased from atcc ) was cultured in 100 ml yt medium ( sambrook et al , loc . cit .) additionally containing 2 % ( w / v ) of glucose and 50 mm sodium phosphate buffer ph 7 . 0 , at 28 ° c . for 36 hours . after harvesting the cells by centrifugation , genomic dna was isolated according to ausubel et al . ( current protocols in molecular biology , volume 1 , greene and john wiley & amp ; sons ( 1994 ), usa ). 100 μg of genomic dna from leuconostoc mesenteroides nrrl - b1355 were partially digested with 0 . 001 units of the restriction enzyme sau3a for 30 minutes , subsequently extracted with phenol : chloroform : isoamyl alcohol . ( 25 : 24 : 1 ) and precipitated with ethanol . 2 . 5 μg of the partially digested dna obtained from leuconostoc mesenteroides nrrl - b1355 were ligated with t4 dna ligase in 1 μg of the bamhl - cut and dephosphorylated vector pbkcmvbamhl ( stratagene ) under the conditions indicated by the manufacturer ( stratagene , pbk phagemid vectors instruction manual & amp ; t4 dna ligase ligation kit ). 2 μl of the ligation mixture were packaged with gigapack iii gold ( stratagene ) according to the instructions of the manufacturer and stored after the amount of phage content had been determined . from the peptide sequences lysc - 66 ( seq . id no . 5 ), lysc - 67 ( seq . id no . 6 ), iysc - 82 ( seq . id no . 7 ), lysc - 83 ( seq . id no . 8 ) and lysc - 88 ( seq . id no . 9 ) obtained after tryptic digestion of the purified alternansucrase protein ( see above ) the peptides lysc - 82 and lysc - 83 , after having undergone reverse translation , were selected for the synthesis of degenerated oligonucleotides ( seq . id no . 10 , seq . id no . 11 ). said oligonucleotides served as primers in a pcr reaction on genomic dna of nrrl - b1355 . all positions within oligonucleotides depicted as n were replaced by inosin in the primer synthesis . the reaction mixture was prepared with the buffers supplied for taq polymerase ( company gibcobrl ). reaction mixture : an 837 bp fragment ( seq . id no . 12 ) resulting from this pcr reaction , the ends of which were blunted with t4 dna polymerase , was cloned into the smal - cut pblueskript vector ( stratagene ). the resulting plasmid was designated palsu - pcr - lysc82 / 83 . after sequencing of the insert and computer - aided translation into the corresponding protein sequences , a data base comparison was carried out in the swiss prot data base . this comparison showed homologies to known glycosyl transferases ( p49331 , p11001 , p68987 , p13470 , p27470 , p29336 ). about 5 , 000 phages of the genomic dna library of leuconostoc mesenteroides nrrl - b1355 were plated out using the bacterial strains and nutrient solutions indicated by the manufacturer ( stratagene ), and after incubation at 37 ° c . for 12 hours were transferred to nitrocellulose filters . this was followed by denaturation of the phages by immersion of the nitrocellulose filters in 1 . 5 m sodium chloride , 0 . 5 m caustic soda solution for 2 minutes and neutralization of the filters by immersion in 1 . 5 m sodium chloride , 0 . 5 m tris - hcl , ph 8 . 0 for 5 minutes . after rinsing the filters in 0 . 2 m tris - hcl , 2 × ssc , the phage dna was bound to the membranes by uv cross link ( stratalinker of the company stratagene , 120 , 000 μj for 30 seconds ). the filters were incubated in a prehybridization solution ( 5 × ssc , 0 . 5 % bsa , 5 × denhardt , 1 % sds , 40 mm sodium phosphate buffer , ph 7 . 2 , 100 mg / l herring sperm - dna , 25 % formamide ) at 42 ° c . for 6 hours . 30 ng of the isolated insert from the plasmid palsu - pcr - lysc82 / 83 were radioactivley labeled by means of a multiprime kit ( boehringer mannheim ) using α - 32 p dctp ( icn biomedicals ). this radioactive probe was added to the prehybridization mixture and the filters were incubated in this hybridization mixture at 42 ° c . overnight . after removal of the hybridization mixture the filters were washed three times in a washing solution ( 0 . 1 × ssc , 0 . 5 % sds ) at 55 ° c . for 15 minutes . an x - ray film ( kodak ) was then placed on the filter for 18 hours . phage colonies , producing hybridization signals , were identified , isolated , resuspended in sm medium and then again plated out in a dissolution such that they could be recognized as single plaques . after these phages were transferred to nitrocellulose filters and subjected to further treatment and hybridization under conditions as described above , hybridizing phages were obtained as individual isolates by means of the radioactive gene probe used . after in vivo excision of the isolated phages in accordance with the manufacturer &# 39 ; s instructions ( stratagene ) the clones as - 19b1 and as - 19b2 could be isolated as plasmids . after complete sequencing of both clones ( agowa ) ( seq . id no . 13 , seq . id no . 14 ) both sequences showed an 1008 bp overlap . the joining of seq . id no . 13 with seq . no . 14 followed by computer aided translation of all possible reading frames allowed a continuous reading frame , starting with the codon atg ( corresponding to the bases 678 to 680 in seq . id no . 1 ), to be identified . as no stop codon could be found in this composed reading frame , additional clones were isolated in order to obtain the complete coding sequence of alternansucrase . therefore , about 5 , 000 phages of the genomic dna library of l . mesenteroides nrrl - b1355 were again examined for hybridization by means of a clone as - 19b2 subfragment radioactively labeled using the multiprime kit ( boehringer mannheim ), as described above . the hybridization probe was prepared with the use of the hindlil ( restriction site in the insert of as - 19b2 )/ sall ( cuts the pbkcmv phagemid vector in the polylinker )- fragment from as - 19b2 . said fragment contains 372 bases of the 3 ′ end of the sequences encoding the above - described reading frame . the screening of the phage library , singling out , and transformation of the phages into plasmids was carried out under the above - described conditions . after complete sequence analysis of the thus isolated clones as - 28b ( see seq . id no . 15 ) and as - 29ba ( seq . id no . 16 ) it was possible to identify an overlap of 960 identical bases ( corresponding to bases 4863 to 5823 in seq . id no . 1 ) between clones as - 19b2 ( seq . id no . 14 ) and as - 28b and an overlap of 567 identical bases ( corresponding to bases 5256 to 5823 in seq . id no . 1 ) between clones as - 19b2 and as - 29ba ( seq . id no . 16 ). clones as - 28b and as - 29ba have 1523 identical bases ( corresponding to bases 5256 to 6779 in seq . id no . 1 ). after computer - aided joining of clones as - 19b1 , as - 19 - b2 and as - 28b a continuous reading frame starting with codon atg ( bases 678 to 680 on the complete sequence ) appeared . this reading frame also does not contain a stop codon . after the joining of clones as - 1 9b1 , as - 1 9b2 , as - 28b and as - 29ba it was possible to identify a reading frame starting with the codon “ atg ” ( corresponding to bases 678 to 680 in seq . id no . 1 ) and ending with “ tm ” ( corresponding to bases 6849 to 6851 in seq . id no . 1 ) encoding 2057 amino acids . in addition to the coding region , the entire isolated and identified dna sequence of the composed clones ( seq . id nos . 13 - 16 ) contains 677 bases in the 5 ′ region and 2469 bases in the 3 ′ region which represent sequences not encoding alternansucrase ( see fig1 ). construction of plasmid palsu - psk for the transformation of e . coli and test of the protein extracts for enzymatic activity plasmids as - 19b1 , as - 19b2 , as - 28b and as - 29ba ( see example 1 ) were joined in the following manner : a notl -( restriction site in the polylinker of vector pbk cmv , company novagen )/ clal - fragment of clone as - 19b1 was inserted into the vector pbluescript sk ( company stratagene ) at the same restriction sites (= first cloning step ). consecutive insertion of the clal / xhol fragment from as - 19b2 , xhol / mlul fragment from as - 28b and mlul / bsabi ( bsabi - cut fragment cloned into the blunted apal restriction site of the vector ) fragment of as - 28b into the clone obtained from the first cloning step produced plasmid palsu - psk ( see fig2 ). this plasmid contains the complete coding sequence of the alternansucrase from leuconostoc mesenteroides nrrl - b1355 as well as non - coding sequences of 677 bp ( promoter region ) in the 5 ′ region and 539 bp in the 3 ′ region ( seq . id no . 17 ). plasmid palsu - psk was then transformed in e . coli ( dh5α company fermentas ). the bacteria were then cultured at 27 ° c . for two days in 50 ml “ terrific broth ” ( the composition of which is described in sambrook et al ., molecular cloning , a laboratory manual , 2 nd edition ( 1989 ) cold spring harbor laboratory press , cold spring harbor , n . y . ( supplemented with 0 . 5 % glucose ) or in a fermentation medium having the following composition : kh 2 po 4 1 . 5 g / l , ( nh 4 ) 2 so 4 5 . 0 g / l , nacl 0 . 5 g / l , na - citrate 1 . 0 g / l , fe 2 + so 4 × 7 h 2 o , 0 . 075 g / l , yeast extract 0 . 5 g / l , tryptone 1 . 0 g / l , glucose 15 . 0 g / l , mgso 4 × 7 , h 2 o 0 . 3 g / l , cacl 2 × 2 h 2 o 0 . 014 g / l , mineral salts 10 ml / l , h 3 bo 3 2 . 5 g / l , cocl 2 × 6 h 2 o 0 . 7 g / l , cuso 4 × 5 h 2 o 0 . 25 g / l , mncl 2 × 4 h 2 o , 1 . 6 g / l , znso 4 × 7 h 2 o 0 . 3 g / l , na 2 moo 4 × 2 h 2 o 0 . 15 g / l , vitamin b1 ( thiamine ) 0 . 005 g / l . all cultures contained 100 mg / l ampicillin . the cells were then harvested by centrifugation , resuspended in 2 ml 50 mm na - phosphate buffer ph 7 . 2 and crushed by a french press . subsequently , they were again subjected to centrifugation to remove solid particles of the crushed cells , and the supernatant ( hereinafter referred to as ( protein ) extract ) was used after sterilfiltration ( sterivex gv 0 . 2 μm , millipore ) for further analyses . for the in vitro preparation of alternan , 200 μl each of the extracts obtained were examined in 2 ml each of 100 mm na - citrate buffer ph 6 . 5 and 20 % ( w / v ) saccharose for activity in the presence and absence of 100 μl of 10 mm maltose . the reaction mixture was incubated at 37 ° c . for 24 hours . in the subsequent precipitation with the same volume of ethanol in the absence of maltose no precipitable polymer was found . in the batch containing maltose , hplc chromatography ( dionex pa - 100 column , running buffer 150 mm naoh , elution buffer 150 mm naoh + 3 m sodium acetate buffer gradient ) showed the formation of oligomers ( see fig3 ). 20 ml each of the individual protein extracts were applied to a 6 % sds - pm gel and separated at a current strength of 20 ma per gel . ( before application to the gels , the extracts were not incubated at 95 ° c .). subsequently , the extracts were examined for sucrase activity according to the method of miller and robyt ( analytical biochemistry 156 ( 1986 ), 357 - 363 ). the control ( dextransucrase nrrl - b - 512f , see example 3 for its preparation ) showed polymerizing activity . the protein extracts of the above - described e . coli cells containing the plasmid palsu - psk , did not show any polymer - forming activity . leuconostoc mesenteroides nrrl - b512f ( obtained from atcc ) was cultured at 28 ° c . for 48 hours in yt - medium ( sambrook et al ., molecular cloning : a laboratory course manual , 2 nd edition ( 1989 ), cold spring harbor press , new york ) additionally containing 1 % of saccharose and 50 mm sodium phosphate buffer ph 7 . 0 . after harvesting the cells by centrifugation , genomic dna was isolated according to ausubel et al . ( current protocols in molecular biology , volume 1 , greene and john wiley & amp ; sons ( 1994 ), usa ). for the recombinant expression of dextransucrase in e . coli , the gene encoding dextransucrase was cloned in the expression vector pet24a ( novagen ) after pcr amplification . for this purpose , an eagl restriction site was introduced at the 5 ′ end of the sequences encoding the dextransucrase and an xhol restriction site at the 3 ′ end , together with the pcr primers used ( 5 ′ b512 - 1 : 5 ′- actgcggccgcatgccatttacagaaaaag - 3 ′; seq . id no . 3 and 3 ′ b512 : 5 ′- actgctcgagttatgctgacacagcatttc - 3 ′; seq . id no . 4 ) derived from the sequence of wo 89 / 12386 . subsequent cloning into the corresponding restriction sites of the polylinker of pet24a was carried out . the resulting plasmid was designated ul5 - 20 . b21 ( de3 ) e . coli cells containing the plasmid ul5 - 20 were cultured in yt medium ( see above ) at 37 ° c . up to an od 600 = 0 . 8 . subsequently , the cells were subjected to induction with 0 . 2 mm iptg and cultured anew at 18 ° c . for 24 hours . after harvesting the cells by centrifugation and resuspending them in sodium phosphate buffer , ph 5 . 2 , the cells were crushed in a french press . the solution obtained was freed from insoluble components by centrifugation and the supernatant containing dextransucrase and referred to hereinafter as the extract was obtained . pcr amplification of the coding region of alternansucrase and cloning in pet24a the coding region of alternansucrase was amplified in a pcr reaction ( see the reaction conditions below ) with genomic dna from the leuconostoc mesenteroides strain nrrl - b1355 as a template . an nhel restriction site was introduced at the 5 ′ end by means of primers a1 - 4 ( seq . id no . 18 ), and a sall - restriction site at the 3 ′ end by means of primer al - 5 ( seq . id no . 19 ). a fragment of about 6200 bp was isolated . a1 - 4 : 5 ′- ggg ccc gct agc atg aaa cm cm gaa aca gt step 4 68 ° c ., 7 minutes ( prolonged by 3 seconds per cycle ) steps 2 to 4 were repeated 35 times altogether before step 5 was carried out . the pcr fragment obtained was purified according to standard methods , treated with the restriction endonucleases nhel and sall , ligated into vector pet24a ( of the company novagen ) which had likewise been cut with these enzymes , and the ligation product was transformed into e . coli . after preparation of the plasmid and restriction digestion , three positive clones were selected . they were designated palsu - pet24a - 3 , palsu - pet24a - 7 and palsu - pet24a - 21 ( see fig4 ), respectively . all contained the sequence indicated in seq . id no . 20 as an insertion . expression of the recombinant alternansucrase in e . coli in shake flask cultures and in the fermenter plasmids palsu - pet24a - 3 , palsu - pet24a - 7 , palsu - pet24a - 21 and pet24a were transformed into e . coli bl21 ( de3 ), of the company novagen , and after initial culturing at 37 ° c . for 3 hours in 3 ml yt medium ( sambrook et al ., molecular cloning , a laboratory manual , 2 nd edition ( 1989 ), cold spring harbor laboratory press , cold spring harbor , n . y .) they were each cultured in shake flasks in 2 replicas in 50 ml davis minimal medium ( difco manual , dehydrated culture media and reagents for microbiology , 10 th edition , detroit mich ., usa ( 1984 )) containing 0 . 2 % glucose instead of dextrose as a carbon source at 37 ° c . until an od 600 of about 0 . 8 was reached . after centrifugation and resuspension , one of the two replica cultures was cultured in davis minimal medium ( dma ) containing 1 % lactose as the carbon source and inductor at 27 ° c . for another 16 hours . the cells of the individual cultures were harvested after centrifugation , resuspended in 50 mm sodium acetate buffer ph 5 . 3 , and a protein extract was prepared as described in example 2 . clone palsu - pet24a - 21 transformed in e . coli bl21 ( de3 ) was cultured in a 2 l fermenter ( biostad b ; b . braun , melsungen ) under the following conditions : fermentation medium : kh 2 po 4 1 . 5 g / l , ( nh 4 ) 2 so 4 5 . 0 g / l , nacl 0 . 5 g / l , na - citrate 1 . 0 g / l , fe 2 + so 4 × 7 h 2 o 0 . 075 g / l , yeast extract 0 . 5 g / l , tryptone 1 . 0 g / l , glucose 15 . 0 g / l , mgso 4 × 7 h 2 o 0 . 3 g / l , cacl 2 × 2 h 2 o 0 . 014 g / l , mineral salts 10 ml / l , h 3 bo 3 2 . 5 g / l , cocl 2 × 6 h 2 o 0 . 7 g / l , cuso 4 × 5 h 2 o 0 . 25 g / l , mncl 2 × 4h 2 o 1 . 6 g / l , znso 4 × 7 h 2 o 0 . 3 g / l , na 2 moo 4 × 2 h 2 o 0 . 15 g / l , vitamin b1 ( thiamine ) 0 . 005 g / l . carbon source : glucose ( 1 . 5 % ( w / v )) is present in the medium , 70 % ( w / v ) glucose solution is added . automatic ph control by ammonia and phosphoric acid at ph 7 . 0 +/− 0 . 1 . a 20 % concentration of po 2 is adjusted in the medium via control by the stirrer . 1 . 5 1 of fermentation medium were inoculated with 50 ml of the preculture . the cells were first cultured at 37 ° c . until the glucose present was consumed . they were then cultured at the same temperature at a feeding rate of 9 g of glucose × l − 1 × h − 1 until an od 600 = 40 was reached . at this time , the temperature of the culture broth was lowered to 20 ° c . and the amount of glucose addition was lowered to 2 g × l − 2 × h − 1 . at a culture temperature of 20 ° c ., the culture was subjected to induction with 0 . 2 mm iptg ( isopropyl - β - d - thiogalactopyranoside ( sigma )). after culturing at 20 ° c . for another 18 hours , the cells were harvested by centrifugation , resuspended in 50 mm sodium phosphate buffer ph 5 . 3 and an extract was prepared as described in example 2 . sds page assay of the activity of the recombinant alternansucrase , periodic acid oxidation and staining according to schiff protein extracts were prepared from e . coli shake flask cultures ( strain bl21 ( de3 )), containing the plasmids palsu - pet24a - 3 , palsu - pet24a - 7 , palsu - pet24a - 21 and pet24a ( control ), respectively . two different extracts were each prepared from the cells transformed with the different extracts , one of said extracts being prepared before induction with iptg and the other one being prepared after induction with iptg at the end of culturing . the activity of these extracts of shake flask cultures ( see example 5 ) was detected by sds page separation of the proteins , followed by sds removal by washing with 50 mm sodium acetate buffer ph 5 . 3 and incubation of the gels in 50 mm sodium acetate ph 5 . 3 , 5 % ( w / v ) saccharose at 37 ° c . for 16 hours , followed by periodic acid oxidation of the polymer formed and staining by means of acidic schiff reagent ( miller and robyt , analytical biochemistry 156 , ( 1986 ), 357 - 363 ). fig5 shows that sucrase activity has not been found for either one of the extracts ( preparation of the extract before and after iptg - induction ) containing the cloning vector pet24a . in the case of strains which had been transformed with the plasmids palsu - pet24a - 3 , palsu - pet24a - 7 and palsu - pet24a - 21 , respectively , all protein extracts showed sucrase activity at the end of the induction phase ( concentrated in one band ). as the polymer formed in the gel can be stained according to the above - described methods by acidic schiff reagent , it can be assumed not to be composed of pure α - 1 , 3 - linked units which would not lead to any staining . as the gene contained in vectors palsu - pet24a - 3 , palsu - pet24a - 7 and palsu - pet24a - 21 , respectively , was isolated from the leuconostoc mesenteroides strain nrrl - b1355 which expresses at least one dextran sucrase apart from alternansucrase , it was not possible to determine unambiguously with this staining method whether the nucleic acid sequence contained in the plasmid actually encodes an alternansucrase . dextrans and alternans can both be detected by this method because both polymers contain α - 1 , 6 linkages . tests for the enzymatic activity of recombinantly prepared alternansucrases after heat treatment and for the specificity of alternansucrase in order to prove polymerization activities , extracts from shake flask cultures were used ( see example 5 ). 100 μl of extract were each added to 2 ml reaction buffer ( 50 mm sodium acetate ph 5 . 3 , 20 % saccharose ) and incubated at 37 ° c . for 24 hours . for comparison , an extract inactivated by a 10 minute treatment at 95 ° c ., and an extract from e . coli bl21 ( de3 ) containing vector pet24a were used . polymer formation was only found in the batch that had not been inactivated , while the batch treated at 95 ° c . for 10 minutes and the batch with the extract from bl21 ( de3 ) containing pet24a did not show any polymer formation . after addition of the same volume of absolute ethanol to all batches , polymers could only be precipitated from the batch which had not been inactivated . this finding is a clear indication of the activity of alternansucrase , because the dextransucrase present in nrrl b - 1355 is inactivated by a treatment at 45 ° c . for 30 minutes , while alternansucrase remains active under these conditions ( lopez - munguia et al ., enzyme microb . technol . 15 ( 1993 ), 77 - 85 ). the enzymatic assay by a coupled enzymatic test of the glucose and fructose released and of the saccharose still contained in the reaction mixture after 24 hours , respectively , revealed that fructose was only present in the extract that was not inactivated . for carrying out the enzymatic test either purified protein or crude protein extract is added in different dilutions to 1 ml batches containing 5 % saccharose and 50 mm acetate , ph 5 . 5 and subjected to incubation at 37 ° c . after 5 minutes , 10 minutes , 15 minutes , 20 minutes , 25 minutes and 30 minutes , 10 μl each are removed from these batches and the enzymatic activity of alternansucrase is terminated by immediate heating to 95 ° c . subsequently , in the coupled photometric test , the portions of fructose and glucose released by alternansucrase and the portion of used - up saccharose , respectively , are determined . for this purpose , 1 μl to 10 μl of the inactivated sample are placed into 1 ml of 50 mm imidazole buffer , ph 6 . 9 , 2 mm mgcl 2 , 1 mm atp , 0 . 4 mm and and 0 . 5 u / ml hexokinase . after sequential addition of about 1 u of glucose - 6 - phosphate dehydrogenase ( from leuconostoc mesenteroides ), about 1 u of phosphoglucose isomerase and about 5 u of invertase , the alteration of adsorption at 340 nm is measured . subsequently , the amount of fructose and glucose released and used - up saccharose , respectively , is calculated according to the lambert - beer law . in control batches ( inactivation of the extract by treatment with 95 ° c . and extract from e . coli containing pet24a ) no significant release of fructose and no decrease of saccharose , respectively , was found in the reaction batch after 24 hours . these results confirm that the specificity of the sucrase encoded by plasmids palsu - pet24a - 3 , palsu - pet24a - 7 and palsu - pet24a - 21 , respectively , is that of a glucosyltransferase . the specificity of a fructosyl transferase , the presence of which has been described for some strains of the genus leuconostoc is to be excluded , because otherwise glucose should have been found . production of alternan by means of alternansucrase prepared in e coli 100 ml of extract obtained by fermentation of e . coli bl21 ( de3 ) containing plasmid palsu - pet24a - 3 ( see example 4 ) were added to 900 ml of reaction buffer ( 50 mm sodium acetate ph 5 . 3 , 20 % saccharose ) and incubated at 37 ° c . for 24 hours . the addition of the same amount of absolute ethanol to the reaction mixture caused the alternan formed to precipitate . after the precipitate was washed twice with 50 % ethanol , it was dried by lyophilization . the yield of dried polymer based on the amount of saccharose used in the reaction was 60 %. 100 mg of the polymer prepared in example 7 and 100 mg of dextran t10 ( pharmacia ) were each dissolved in 1 ml of water . 40 μl each of these solutions were added to 700 μl reaction buffer ( 50 mm potassium phosphate ph 5 . 7 , 8 units of dextranase , icn biomedicals inc . no . 190097 ), and incubated at 37 ° c . for 16 hours . 50 μl of the polymer solutions not treated with dextranase ( see fig6 ) and 50 μl of the polymer solutions treated with dextranase ( fig7 ) were analyzed by hplc ( dionex , column pa - 100 , naoh / naoh - naac gradient ). in the case of dextran t10 the cleavage of the polymer into different molecules of lower molecular weights can be clearly seen . the entire high molecular weight dextran is converted by dextranase into smaller units ( mostly isomaltose ). by contrast , in the case of alternan , short chained oligosaccharides only appear in small amounts after dextranase incubation . most of the alternan is not digestible by dextranase . this finding suggests that the product prepared by recombinant alternansucrase is not dextran , but alternan which is known to be hardly accessible to enzymatic digestion by dextranase ( lopez - mungia et al ., enzyme microb . technol . 15 , ( 1993 ), 77 - 85 ). 100 μl extract from shake flask cultures ( see example 5 ) were added to 2 ml of reaction buffer ( 50 mm sodium acetate , ph 5 . 3 , 20 % saccharose ). 50 units of dextranase ( biomedicals inc . no . 190097 ) were additionally added to another batch . two corresponding batches which contained dextransucrase from leuconostoc mesenteroides nrrl - b512f instead of the enzyme extract served as controls ; one of these two batches had dextranase additionally admixed to it . after precipitation with ethanol , the reaction batch with dextransucrase and dextranase did not show any polymer formation . all other batches were found to show polymer formation . oligoalternan was prepared as described in example 2 , with a protein extract in the presence of maltose and was subsequently detected ( see fig8 ) by hplc - chromatography ( see example 2 ). for comparison , a portion of this batch was admixed with 50 units of dextranase ( biomedicals inc . 190097 ) after preparation of oligoalternan and subsequently separation by hplc chromatography was carried out as well ( see fig9 ). a comparison of the two chromatograms shows that not only the height of the two peaks which can be allocated to the oligoalternan ( α and β - anomer ) ( retention time between 15 . 87 and 16 . 61 minutes ) but also the height of all the other peaks , the first signs of which are already visible without dextranase , remain unchanged . this finding suggests that recombinantly prepared alternansucrase allows oligoalternan to be prepared without the simultaneous production of oligodextran . oligodextran would be liable to digestion by dextranase , which would have to show up in a decrease of the height of the peaks in the hplc chromatogram , if oligodextran were present . in order to further analyze the alternan produced in vitro a methylation analysis was carried out : the permethylation was performed as described by ciucanu and kerek ( carbohydr . res . 131 ( 1984 ), 209 - 218 ) by using naoh / mel in dmso or by using a modified method according to hakomori ( journal of biochemistry 55 ( 1964 feb ), 205 - 208 ) which relies on the use of freshly prepared li - dimsyl / mel ( dimsyl = methylsulfinyl carbanion ) in dmso at room temperature . all reactions are performed under a nitrogen atmosphere . the permethylation products are isolated by extracting the excess of methyliodide by the use of dichlormethan . dmso and salts were washed out at the end . the permethylated glucans were hydrolyzed with 2n trifluorine acetic acid at 120 ° c . for 1 - 3 hours . after cooling the acid was removed by nitrogen . then the resulting glucans were co - distilled with a small amount of toluene , afterwards reduced by nabd 4 in 1n ammonia and finally , acetylated by pyridine / acetanhydrid ( 3 h , 90 ° c .). the products were extracted by dichlormethan and washed with nahco 3 . the products in the organic phase were analyzed by gas chromatography . the acetylated products were analyzed by gas chromatography which was performed with a chromatograph manufactured by the carlo - erba company model gc 6000 vega equipped with an on - column injector , a 25 m cpsol8cb and a fid - detector . as a carrier gas hydrogen ( 80 kpa ) was used . the identification and integration of the peaks was performed as described by sweet et al . ( carbohydr . res . 40 ( 1975 ), 217 ). furthermore , small amounts ( rel . amount 0 . 2 - 0 . 4 mol %) of the following components were also found : 1 , 4 , 5 - and 1 , 3 , 4 , 5 - sorbit and another tetraacetyl component ( 1 , 5 , x , y ). it is supposed that these components are due to incomplete methylation . the following amounts were found for the above mentioned components in different experiments which were performed by changing the length of hydrolysis ( indicated in bold by the number of hours ) ( ma = methylation analysis1 ; ma - b = methylation analysis 2 ): construction of an expression cassette for plants : vacuolar and plastidic expression of an alternansucrase by using plasmid alsu - pet24a as a template and the pcr primers al - 5 ′- 1 . 2 and al - 3 ′- 2 . 2 ( see seq id no 53 and 54 ) we amplified the coding region of alternansucrase from leuconostoc mesenteroides which was then cut by the restriction enzymes sall and pstl . afterwards the resulting fragments were cloned into sall and sdal digested plasmids a ) pbinar - pat - hyg and b ) pbinar - fnr - hyg . the resulting plasmids were called a ) pat - alsu - hyg ( see fig1 ) and b ) fnr - alsu - hyg ( see fig1 ). note : the bacterial secretion signal peptide was removed from the cds by choice of the pcr primers . the steps 2 to 4 were repeated 35 times in a cyclical manner . leaves or tubers from potato plants transformed via agrobacteria with plasmids pat - alsu - hyg and fnr - alsu - hyg , respectively , were pulverized in a mill , type mm 200 , ( retsch gmbh & amp ; co . kg , 42781 haan , germany ) at 30 hz for 50 sec . rna was extracted according to logemann et al . ( anal . biochem . 163 ( 1987 ), 16 - 20 ). 50 μg rna per sample were loaded on 1 % agarose gels containing formaldehyde . after electrophoresis the rna was transferred to nylon membranes ( hybond n , amersham , uk ) by the capillary transfer method ( sambrook et al ., molecular cloning : a laboratory manual , 2nd issue ; cold spring harbor laboratory press , ny , usa ( 1989 )). fixation of nucleic acids at the membrane was achieved by uv crosslinking ( stratalinker by stratagene ). membranes were prehybridized at 42 ° c . in hybridization buffer ( 25 % ( v / v ) formamide , 250 mm sodium phosphate , ph 7 . 2 , 250 mm sodiumchloride , 1 mm edta 7 % ( w / v ) sds , 25 % ( w / v ) polyethyleneglycol 6000 , 0 , 25 mg / ml sheared salmon sperm dna ) for 6 h . afterwards hybridization was performed at 42 ° c . over night in hybridization buffer containing a radiolabelled probe in addition . the radioactive probe was prepared by using the random primed dna labelling kit ( boehringer mannheim , 1004760 ) and the approx . 4 kb kpnl / xhol - fragment from plasmid palsu - psk according to the manufacturers manual . membranes were washed at 50 ° c . once for 20 min in 3 × ssc ( sambrook et al ., molecular cloning : a laboratory manual , 2nd issue ; cold spring harbor laboratory press , ny , usa ( 1989 )) followed by washing once for 20 min in 0 . 5 × ssc before exposing the membrane to an x - ray - film over night . gaaagggaga ataatta atg aaa caa caa gaa aca gtt acc cgt aaa aaa 710 ctt tat aaa tcc ggt aag gtt tgg gtt gca gca gct act gca ttt gcg 758 gta ttg ggg gtt tca act gta aca aca gtc cat gcg gat aca aat tcg 806 aat gtc gct gtt aag caa ata aat aat aca gga acc aat gat tct ggc 854 gaa aaa aag gta ccg gtt cca tca act aat aat gat agt ttg aag caa 902 gga aca gat ggt ttt tgg tat gat tca gac ggc aat cgt gtc gat cag 950 gly thr asp gly phe trp tyr asp ser asp gly asn arg val asp gln aag acc aat cag att ctg ctt act gcg gaa caa ctt aaa aaa aat aac 998 gaa aaa aat tta tca gta atc agt gat gat aca tca aaa aaa gat gat 1046 gaa aat att tct aag cag acc aaa att gct aat caa caa aca gta gat 1094 act gct aaa ggc ctg act acc agt aat tta tct gat ccc atc act ggg 1142 ggt cac tat gaa aat cac aat ggc tac ttt gtt tat ata gat gct tca 1190 gly his tyr glu asn his asn gly tyr phe val tyr ile asp ala ser gga aaa caa gta aca ggt ttg caa aat att gat ggt aat tta caa tat 1238 ttt gat gac aat gga tat caa gtc aag gga tcc ttc cga gat gtc aac 1286 ggc aag cat atc tat ttt gat tca gta aca ggg aaa gct agt tca aat 1334 gly lys his ile tyr phe asp ser val thr gly lys ala ser ser asn gtt gat att gtt aac ggt aaa gct caa gga tat gat gcg caa ggc aac 1382 caa tta aag aaa agt tat gtc gcc gat agt tct ggg caa act tac tat 1430 ttt gat ggt aat ggc caa ccg tta atc ggc ttg caa aca att gat ggg 1478 aac cta caa tat ttt aac caa caa ggg gtt caa ata aag ggt ggt ttc 1526 caa gat gtt aac aat aaa cgt att tat ttt gca cca aac aca ggt aat 1574 gln asp val asn asn lys arg ile tyr phe ala pro asn thr gly asn gcc gtt gcc aat act gaa ata att aac ggt aaa tta cag ggg cgt gac 1622 ala val ala asn thr glu ile ile asn gly lys leu gln gly arg asp gca aat ggt aac cag gta aag aat gca ttt agt aaa gat gtt gca gga 1670 aat aca ttt tat ttt gac gca aac ggt gtg atg tta aca ggg ttg caa 1718 asn thr phe tyr phe asp ala asn gly val met leu thr gly leu gln act att tca gga aag aca tat tat ctt gat gaa caa gga cac ctg aga 1766 thr ile ser gly lys thr tyr tyr leu asp glu gln gly his leu arg aaa aat tac gcg gga aca ttc aat aat cag ttt atg tac ttc gat gct 1814 gat aca ggt gcg ggt aaa aca gcg att gaa tat caa ttt gat caa gga 1862 ttg gta tca caa agt aat gaa aat act cct cac aat gcc gca aag tct 1910 leu val ser gln ser asn glu asn thr pro his asn ala ala lys ser tat gat aaa agt agt ttt gaa aat gtt gat ggt tac tta aca gca gat 1958 tyr asp lys ser ser phe glu asn val asp gly tyr leu thr ala asp aca tgg tat cgt cca acc gat att tta aaa aat gga gat act tgg acg 2006 thr trp tyr arg pro thr asp ile leu lys asn gly asp thr trp thr gca tct acc gaa act gat atg cgt ccg ctt tta atg aca tgg tgg cct 2054 gac aaa caa aca caa gca aat tac ttg aat ttt atg tct agt aaa gga 2102 asp lys gln thr gln ala asn tyr leu asn phe met ser ser lys gly ctt ggt ata acg acc act tat aca gca gct acg tca caa aaa aca cta 2150 aat gac gca gcc ttt gtt att caa aca gca att gaa caa caa ata tct 2198 ttg aaa aaa agt act gag tgg tta cgt gat gca att gat agt ttt gtg 2246 leu lys lys ser thr glu trp leu arg asp ala ile asp ser phe val aag acg caa gct aat tgg aat aag caa aca gaa gat gaa gct ttc gat 2294 ggt ttg cag tgg ctt caa ggg gga ttc cta gct tat caa gat gat tca 2342 cat cgg acg ccg aat act gat tca gga aat aac aga aaa cta gga cgt 2390 caa cca att aat atc gat ggt tcg aaa gat aca act gat ggt aaa ggc 2438 tct gaa ttc tta tta gct aac gat att gac aac tca aat ccg att gtt 2486 caa gct gag caa tta aac tgg cta cac tat tta atg aat ttt ggt agt 2534 gln ala glu gln leu asn trp leu his tyr leu met asn phe gly ser att aca ggt aat aat gac aat gcg aat ttt gat ggc att cgt gta gat 2582 gct gtt gat aat gtt gat gct gat tta cta aaa ata gct ggc gat tat 2630 ttt aaa gct cta tat ggt aca gat aaa agc gac gcc aat gcc aat aag 2678 cat ttg tct att tta gaa gac tgg aac ggt aaa gat cct cag tat gtt 2726 his leu ser ile leu glu asp trp asn gly lys asp pro gln tyr val aat caa cag ggc aat gcg caa tta aca atg gat tac aca gtt act tca 2774 asn gln gln gly asn ala gln leu thr met asp tyr thr val thr ser cag ttt ggc aat tct cta aca cat ggc gcc aac aac agg agt aac atg 2822 gln phe gly asn ser leu thr his gly ala asn asn arg ser asn met tgg tat ttc tta gat act ggc tat tat ctt aat gga gat ctt aat aag 2870 aag ata gta gat aag aac cgt cca aat tct ggc act ttg gtt aac aga 2918 lys ile val asp lys asn arg pro asn ser gly thr leu val asn arg att gct aat tca ggt gat aca aaa gtt att cca aat tat agt ttt gtt 2966 ile ala asn ser gly asp thr lys val ile pro asn tyr ser phe val aga gca cat gat tac gat gct caa gat cca att aga aaa gcc atg att 3014 gat cat ggt att att aaa aac atg cag gat act ttc act ttt gac caa 3062 ctg gct cag gga atg gaa ttc tac tat aaa gat caa gag aat ccg tct 3110 leu ala gln gly met glu phe tyr tyr lys asp gln glu asn pro ser ggt ttc aaa aag tat aac gat tat aac tta cct agt gct tat gca atg 3158 gly phe lys lys tyr asn asp tyr asn leu pro ser ala tyr ala met ttg ttg act aat aag gat act gta cct cgt gtc tat tat gga gat atg 3206 leu leu thr asn lys asp thr val pro arg val tyr tyr gly asp met tac ctc gaa ggc ggg caa tat atg gaa aaa ggg acg att tac aat cct 3254 tyr leu glu gly gly gln tyr met glu lys gly thr ile tyr asn pro gtc att tca gcg ttg ctc aaa gct aga ata aaa tat gtt tct ggt ggg 3302 caa aca atg gct acc gat agt tct gga aaa gac ctt aaa gat ggc gaa 3350 act gat ttg tta aca agt gtt cga ttt ggt aaa gga att atg aca tca 3398 thr asp leu leu thr ser val arg phe gly lys gly ile met thr ser gat caa acc aca aca caa gac aat agc caa gat tat aaa aat caa ggc 3446 atc ggt gtc att gtt ggt aat aac cct gac ctt aag ttg aac aat gat 3494 aag acc att acc ttg cat atg gga aag gcg cat aag aat caa ctt tac 3542 lys thr ile thr leu his met gly lys ala his lys asn gln leu tyr cgt gcc tta gta tta tca aat gac tca gga att gat gtt tat gat agt 3590 gat gat aaa gca cca act ttg aga aca aat gac aac ggt gac ttg att 3638 ttc cat aag aca aat acg ttt gtg aag caa gat gga act att ata aat 3686 tac gaa atg aag gga tca tta aat gct tta att tca ggt tat tta ggt 3734 gtc tgg gtg cca gtt gga gct agt gat tca caa gat gct cgt aca gtg 3782 gca act gag tca tca tca agt aat gat ggt tct gta ttc cat tca aat 3830 gct gca tta gat tct aat gtt ata tat gaa ggc ttt tca aac ttt caa 3878 ala ala leu asp ser asn val ile tyr glu gly phe ser asn phe gln gcg atg ccg act tct cct gag caa agt aca aat gtt gtt att gca aca 3926 aag gct aac tta ttt aaa gaa tta ggt att act agt ttt gag tta gca 3974 cct caa tat agg tct agt ggt gac act aat tac ggt ggc atg tca ttc 4022 pro gln tyr arg ser ser gly asp thr asn tyr gly gly met ser phe tta gat tct ttc tta aat aat ggt tat gca ttt acc gat aga tat gat 4070 tta ggc ttt aac aaa gca gac ggg aat cct aac cca aca aag tat gga 4118 aca gat caa gat tta cgt aat gca ata gag gca tta cac aaa aac ggc 4166 thr asp gln asp leu arg asn ala ile glu ala leu his lys asn gly atg cag gct ata gct gat tgg gtt cct gac caa ata tat gct tta cca 4214 gga aag gaa gtt gtt acc gct act aga gta gac gaa cgg gga aat caa 4262 cta aaa gac aca gat ttt gtc aac tta ctc tat gtt gct aat act aaa 4310 agt agt ggt gtg gat tat cag gca aag tat ggc ggc gaa ttt tta gat 4358 ser ser gly val asp tyr gln ala lys tyr gly gly glu phe leu asp aaa tta aga gaa gag tac cca tcg tta ttc aaa cag aac caa gta tcg 4406 lys leu arg glu glu tyr pro ser leu phe lys gln asn gln val ser aca ggt cag cca att gat gct tct aca aaa att aag caa tgg tca gct 4454 aaa tat atg aat ggg acc aat att tta cat cga ggt gct tat tat gtt 4502 lys tyr met asn gly thr asn ile leu his arg gly ala tyr tyr val ttg aaa gac tgg gct act aac cag tat ttt aac att gca aaa acg aat 4550 leu lys asp trp ala thr asn gln tyr phe asn ile ala lys thr asn gaa gta ttt ttg cca cta cag ttg cag aat aaa gat gcg caa act ggt 4598 glu val phe leu pro leu gln leu gln asn lys asp ala gln thr gly ttc att agt gat gcc tcc ggt gta aaa tat tac tca att agt ggt tat 4646 caa gca aaa gat act ttt att gaa gat ggt aat ggg aat tgg tat tac 4694 gln ala lys asp thr phe ile glu asp gly asn gly asn trp tyr tyr ttt gat aaa gat ggt tac atg gtg cgt tcg cag caa gga gaa aat cct 4742 phe asp lys asp gly tyr met val arg ser gln gln gly glu asn pro ata aga aca gtc gaa act agt gtc aac aca cga aac ggt aat tat tac 4790 ttt atg cca aat ggt gtc gag ttg cgc aaa ggc ttt gga acg gat aat 4838 phe met pro asn gly val glu leu arg lys gly phe gly thr asp asn agt ggt aat gtc tat tat ttt gat gat caa ggt aag atg gtg aga gat 4886 ser gly asn val tyr tyr phe asp asp gln gly lys met val arg asp aaa tac att aac gat gat gct aat aat ttt tat cac tta aat gtt gat 4934 ggg act atg tct cga gga cta ttt aaa ttt gat tct gat act cta cag 4982 tat ttt gct agt aat ggt gtc caa ata aaa gat agt tat gcg aag gat 5030 tyr phe ala ser asn gly val gln ile lys asp ser tyr ala lys asp agt aaa ggc aat aaa tat tat ttt gac tca gct aca gga aat aac gat 5078 act ggg aaa gcc caa act tgg gat ggt aat ggc tac tat att act att 5126 gat tct gat gcg aac aat aca att ggg gtt aac aca gac tac act gcc 5174 tac atc act agc tcg ctg cgc gaa gat ggc tta ttt gct aac gca cct 5222 tyr ile thr ser ser leu arg glu asp gly leu phe ala asn ala pro tac ggt gtt gta aca aaa gac caa aat ggt aac gat ctt aag tgg cag 5270 tat att aac cat acg aaa cag tac gaa ggg caa caa gtg caa gtc acg 5318 cgt caa tac aca gac agt aag gga gtc agc tgg aac tta att acc ttt 5366 arg gln tyr thr asp ser lys gly val ser trp asn leu ile thr phe gct ggt ggt gat tta caa gga caa agg ctt tgg gtg gat agt cgt gcg 5414 tta act atg aca cca ttt aaa acg atg aac caa ata agc ttc att agt 5462 tat gct aac cgc aat gat ggg ttg ttt ttg aat gcg cca tac caa gtc 5510 tyr ala asn arg asn asp gly leu phe leu asn ala pro tyr gln val aag ggg tat caa tta gct ggg atg tcc aac caa tac aag ggc caa caa 5558 gtg acc att gct ggg gtg gcg aac gtt tct gga aaa gac tgg agt ctg 5606 val thr ile ala gly val ala asn val ser gly lys asp trp ser leu att agt ttt aat ggg aca cag tac tgg att gat agt cag gca ttg aat 5654 ile ser phe asn gly thr gln tyr trp ile asp ser gln ala leu asn acc aat ttc aca cat gac atg aac caa aag gtc ttt gtc aat aca act 5702 agt aat ctt gat ggg tta ttc tta aat gcg cca tac cgt caa ccg ggt 5750 ser asn leu asp gly leu phe leu asn ala pro tyr arg gln pro gly tat aag tta gcc ggt ttg gct aaa aat tac aac aac caa acg gtt act 5798 gtt agt caa cag tac ttt gat gat caa ggc acg gtc tgg agt cag gtt 5846 gtc ctt ggg ggt cag acg gtc tgg gtt gat aac cat gca ttg gca cag 5894 atg caa gtt agt gat aca gac caa cag ctc tat gtg aat agc aat ggt 5942 cgg aat gat ggg tta ttc ttg aat gcg cca tat cgt ggt caa ggg tca 5990 arg asn asp gly leu phe leu asn ala pro tyr arg gly gln gly ser caa ctg ata ggc atg acg gca gat tat aat ggg caa cat gta caa gtg 6038 gln leu ile gly met thr ala asp tyr asn gly gln his val gln val acc aag caa ggg caa gat gcc tat ggt gca caa tgg cgt ctt att acg 6086 thr lys gln gly gln asp ala tyr gly ala gln trp arg leu ile thr cta aat aat caa cag gtc tgg gtt gat agt cgc gct ttg agc aca aca 6134 atc atg caa gcc atg aat gat aat atg tat gta aat agc agc caa cgg 6182 aca gat ggc ttg tgg tta aac gca cct tat acg atg agt ggg gct aaa 6230 thr asp gly leu trp leu asn ala pro tyr thr met ser gly ala lys tgg gct ggt gat aca cgt tca gct aat ggg cgc tat gtc cat att tca 6278 trp ala gly asp thr arg ser ala asn gly arg tyr val his ile ser aaa gct tat tca aac gaa gtc ggc aat aca tat tac ttg acg aat ttg 6326 aat ggt caa agc aca tgg att gac aag cgg gcg ttt act gtg acc ttc 6374 asn gly gln ser thr trp ile asp lys arg ala phe thr val thr phe gat cag gtg gtg gca tta aat gca acg att gtg gca cgc caa cga cca 6422 gat ggg atg ttt aag aca gca cca tat ggt gaa gcg ggg gcg cag ttt 6470 asp gly met phe lys thr ala pro tyr gly glu ala gly ala gln phe gtc gat tat gtg aca aac tat aac cag caa acc gtg cca gta aca aag 6518 caa cat tca gat gct cag ggg aat caa tgg tac tta gcg aca gtg aat 6566 gln his ser asp ala gln gly asn gln trp tyr leu ala thr val asn ggg aca caa tac tgg att gat caa cgg tca ttt tca cca gta gta acg 6614 gly thr gln tyr trp ile asp gln arg ser phe ser pro val val thr aag gtg gtt gat tat caa gct aag att gtg cca cgg aca aca cgt gat 6662 ggt gtg ttt agt ggc gca ccc tat ggg gaa gtg aat gct aag cta gtt 6710 gly val phe ser gly ala pro tyr gly glu val asn ala lys leu val aac atg gca act gcg tat caa aat caa gtt gtc cat gcg aca ggg gaa 6758 tat acg aat gct tca ggg atc aca tgg agt cag ttc gcg tta agc ggg 6806 tyr thr asn ala ser gly ile thr trp ser gln phe ala leu ser gly caa gaa gac aag cta tgg att gat aag cgt gct ttg caa gct 6848 met lys gln gln glu thr val thr arg lys lys leu tyr lys ser gly gln ile asn asn thr gly thr asn asp ser gly glu lys lys val pro val pro ser thr asn asn asp ser leu lys gln gly thr asp gly phe trp tyr asp ser asp gly asn arg val asp gln lys thr asn gln ile thr thr ser asn leu ser asp pro ile thr gly gly his tyr glu asn his asn gly tyr phe val tyr ile asp ala ser gly lys gln val thr tyr gln val lys gly ser phe arg asp val asn gly lys his ile tyr lys arg ile tyr phe ala pro asn thr gly asn ala val ala asn thr asp ala asn gly val met leu thr gly leu gln thr ile ser gly lys thr tyr tyr leu asp glu gln gly his leu arg lys asn tyr ala gly lys thr ala ile glu tyr gln phe asp gln gly leu val ser gln ser phe glu asn val asp gly tyr leu thr ala asp thr trp tyr arg pro thr asp ile leu lys asn gly asp thr trp thr ala ser thr glu thr ala asn tyr leu asn phe met ser ser lys gly leu gly ile thr thr glu trp leu arg asp ala ile asp ser phe val lys thr gln ala asn trp asn lys gln thr glu asp glu ala phe asp gly leu gln trp leu gln gly gly phe leu ala tyr gln asp asp ser his arg thr pro asn thr asp ser gly asn asn arg lys leu gly arg gln pro ile asn ile asn trp leu his tyr leu met asn phe gly ser ile thr gly asn asn ala gln leu thr met asp tyr thr val thr ser gln phe gly asn ser leu thr his gly ala asn asn arg ser asn met trp tyr phe leu asp asp thr lys val ile pro asn tyr ser phe val arg ala his asp tyr gln tyr met glu lys gly thr ile tyr asn pro val ile ser ala leu leu lys ala arg ile lys tyr val ser gly gly gln thr met ala thr ser val arg phe gly lys gly ile met thr ser asp gln thr thr thr his met gly lys ala his lys asn gln leu tyr arg ala leu val leu thr phe val lys gln asp gly thr ile ile asn tyr glu met lys gly asn val ile tyr glu gly phe ser asn phe gln ala met pro thr ser pro glu gln ser thr asn val val ile ala thr lys ala asn leu phe lys glu leu gly ile thr ser phe glu leu ala pro gln tyr arg ser arg asn ala ile glu ala leu his lys asn gly met gln ala ile ala asp trp val pro asp gln ile tyr ala leu pro gly lys glu val val thr ala thr arg val asp glu arg gly asn gln leu lys asp thr asp phe val asn leu leu tyr val ala asn thr lys ser ser gly val asp tyr pro ser leu phe lys gln asn gln val ser thr gly gln pro ile asp ala ser thr lys ile lys gln trp ser ala lys tyr met asn gly thr asn ile leu his arg gly ala tyr tyr val leu lys asp trp ala thr asn gln tyr phe asn ile ala lys thr asn glu val phe leu pro leu gln leu gln asn lys asp ala gln thr gly phe ile ser asp ala tyr met val arg ser gln gln gly glu asn pro ile arg thr val glu val glu leu arg lys gly phe gly thr asp asn ser gly asn val tyr tyr phe asp asp gln gly lys met val arg asp lys tyr ile asn asp asp ala asn asn phe tyr his leu asn val asp gly thr met ser arg gly leu phe lys phe asp ser asp thr leu gln tyr phe ala ser asn leu arg glu asp gly leu phe ala asn ala pro tyr gly val val thr lys asp gln asn gly asn asp leu lys trp gln tyr ile asn his thr ser lys gly val ser trp asn leu ile thr phe ala gly gly asp leu gln gly gln arg leu trp val asp ser arg ala leu thr met thr pro phe lys thr met asn gln ile ser phe ile ser tyr ala asn arg asn asp gly leu phe leu asn ala pro tyr gln val lys gly tyr gln leu ala gly met ser asn gln 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