Patent Application: US-201514625916-A

Abstract:
the present invention relates to the use of transgenic plants for the expression of vitamin b12 binding proteins . plant cells are transformed with nucleotide sequences adapted for expression and secretion of vitamin b12 binding proteins . the present invention also relates to the use of recombinant vitamin b12 binding proteins from plants in analytical tests and treatment of vitamin b12 deficiency . also disclosed is a method for purification of recombinant vitamin b12 binding proteins .

Description:
as shown in fig1 , the extensin signal peptide encoding nucleotide sequence from arabidopsis thaliana was fused to the nucleotides encoding mature human intrinsic factor ( fig2 ). this construct was inserted in the plant transformation vector crc179 . the vector ppzp 211 ( hajdukiewicz , p ; svab , z ; & amp ; maliga , p . 1994 plant mol . biol . 25 , 989 - 994 ) was digested with ecori and kpni and a panos sequence was released from pgptv kan ( becker , d ; kemper , e ; schell , j & amp ; masterson , r . 1992 plant mol biol 20 , 1195 - 1197 ) by the same set of enzymes and cloned into the ppzp 211 vector . the resulting vector was digested with psti and kpni and blunt - ended . a blunt - ended ecori and hindiii fragment containing the 35s camv promoter from the vector described in jefferson , r a ; kavanagh , t a & amp ; bevan , m w . 1987 embo j . 6 , 3901 - 3907 was cloned into the blunt - ended vector . this vector was named crc - 179 . the bacteria agrobacterium tumefaciens was transformed with this recombinant vector . the agrobacterium tumefaciens strain used was gv3101 ( pmp90 ) ( koncz and schell , 1986 ) carrying the binary plasmid crc - 179 with an insert encoding a cbp cloned into the xbai - xmai sites . the insert sequences are shown in the figures : 1 , 3 , 4 , & amp ; 5 . the bacteria were grown to stationary phase in 200 ml liquid culture at 28 - 30 c , 250 rpm in sterilized lb media ( 10 g tryptone , 5 g yeast extract , 5 g nacl per liter water ) carrying added rifampicin ( 100 mg / ml ), streptomycin ( 100 mg / ml ) and gentamycin ( 50 mg / ml ). cultures were started from a 1 : 200 dilution of a smaller overnight culture and grown for 16 - 18 hours . bacteria cells were harvested by centrifugation for 10 min at 5500 g at room temperature and then resuspended in 400 ml inoculation medium ( 10 mm mgcl2 , 5 % w / v sucrose and 0 . 05 % v / v silwet l - 77 ( lehle seeds , round rock , tex ., usa )). the recombinant a . tumefaciens bacteria were used to transform arabidopsis thaliana plants . the arabidopsis plants were transformed by the floral dip method ( clough and bent , 1998 ). arabidopsis plants ( ecotype col - 0 ) were grown to flowering stage in growth chamber , 20 c day / 18 c night with licl lighting for 18 h / day , humidity 70 %. between 20 and 25 plants were planted per 64 cm2 pot in moistened soil mixture consisting of : 40 kg soil orange and 40 kg soil green ( stenrgel mosebrug a / s kjellerup , dk ), 25 liter 4 - 8 mm fibroklinker ( optiroc , randers , dk ), 12 liter vermiculite ( skamol , dk ) and 300 g osmocote plus npk 15 - 5 - 11 , 3 - 4 months ( scott &# 39 ; s , uk ). to obtain more floral buds per plant , inflorescences were clipped after most plants had formed primary bolts , relieving apical dominance and encouraging synchronized emergence of multiple secondary bolts . plants were dipped when most secondary inflorescences were about 7 - 13 cm tall ( 7 - 9 days after clipping ). the transgenic agrobacterium suspension was added to a 400 ml beaker and plants were inverted into the suspension such that all above - ground tissues minus the rosette were submerged . the plants were removed after 10 - 15 sec of gentle agitation and placed in horizontal position in a sealed plastic bag for 24 hours at room temperature . after 24 hours the plants were moved to the growth camber and the plastic bags were removed . plants were grown 3 - 4 weeks until siliques were brown and dry . seeds were harvested and allowed to dry at room temperature for 7 days . seed were surface sterilized by a treatment with 0 . 5 % sodium hypochlorite containing 0 . 05 % v / v tween 20 for 7 min , then with 70 % ethanol for 2 min , followed by three rinses with sterile water . to select for transformed plants the sterilized seeds were plated on kanamycin selection plates at a density of approximately 2000 seeds per 144 cm2 and grown for 8 - 10 days at 21 c under light for 16 hours per day . selection plates contained 1 × ms medium ( duchefa , haarlem , nl # m 0222 ), 1 % w / v sucrose , 0 . 9 % w / v agar noble ( difco , detroit , usa ), 50 mg / ml kanamycin , ph 5 . 7 . after selection the transformed plants were transferred to growth chambers ( see plant growth ). seeds from these infected plants were planted and recombinant plants were identified by western - blotting analysis . seeds from recombinant plants were used to grow new recombinant plants called if - plants . one kilogram of three week old if - plants was homogenization with 2 liters of phosphate buffer and clarified by centrifugation . this extract contained 100 mg recombinant human if with cbc . this if had specificity for cobalamin binding whereas the analog cobinamid was not bound by the if as tested by the method described by ( 15 ). fig7 shows that the rhif from plants and native human gastric if have the same specificity for binding cbl , but not cobinamid . the transgenic plants were analyzed as described by ( 16 ) and shown to contain no cobalamin . this shows that the plant rhif was at the apo - form . analysis of the protein from these transgenic plants showed that they express a protein of approximately 50 kda as recognized on a western - blot with antibodies against human if ( see fig8 , 10 ). amino acid sequencing of the n - terminal region of this 50 kda protein showed the same sequence as that present in mature natural if ( fig2 ). therefore , the post - translational cleavage of the extensin signal peptide from the fusion protein results in the secretion of a recombinant if with the correct n - terminus . the size of approximately 50 kda indicate that the protein was full - length . a mature full - length if is 399 amino acids with a calculated molecular weight of 43412 da . the recombinant if contains some carbohydrates as shown by pas - staining of blotted recombinant protein separated by sds - page ( see fig9 ). deglycosylation of rhif from plants results in a protein with an approximately similar observed and calculated molecular weight ( fig8 ). the molecular size of natural human if was approximately 5 kda larger than the recombinant plant if as estimated from the western - blot . some difference in the carbohydrate composition is expected between natural human if and recombinant plant if since carbohydrate composition is tissue specific and to some extent unique to each individual . differences in the molecular weight between natural and recombinant if may therefore be a result of different carbohydrate composition . this was shown by removal of the carbohydrates from human gastric if , human if expressed in yeast and human if expressed in plants . the deglycosylated form of these three if proteins to have the same molecular weight ( see fig8 ). on a western - blot containing purified rhif from transgenic arabidopsis thaliana , two minor bands of approximately 30 and 20 kda were observed in addition to the 50 kda band with mature rhif ( fig1 ). n - terminal sequencing of these two bands showed , that the 30 kda band contained the n - terminal sequence of mature human if . the 20 kda band contained an n - terminus located at glycine - 285 of mature if . the calculated molecular weight of the fragment containing amino acid residues 1 - 284 was 30612 da and the fragment containing amino acid residues 285 - 399 had a calculated molecular weight of 12817 da . the inconsistency between the observed 20 kda and the calculated 12817 da most likely was a result of glycosylation of one or more of the four potential n - linked glycosylation sites of the fragment containing amino acid residue 285 - 399 . the pas - staining also recognized the 20 kda band showing that some glycosylation was present on the fragment . no pas - staining was observed for the 30 kda fragment although one potential n - linked glycosylation site was present on this fragment . this is consistent with that no difference was found between the observed and calculated molecular weight for the fragment containing amino acid residues 1 - 284 . fig1 shows that rhif from plants in complex with vitamin b12 binds to the human intestinal receptor protein cubilin . the apo - form of rhif does not bind to the cubulin receptor . for comparision binding of human gastric if and hog if were also shown to bind to the cubulin receptor only when in holo - form . these results show , that recombinant human intrinsic factor from plants behaves as native gastric intrinsic factor with respect to receptor binding . arabidopsis thaliana was transformed with a vector construct which contained a nucleotide sequence encoding the signal peptide from the phaseolus vulgaris chitinase ch5b fused to the nucleotide sequence encoding mature human intrinsic factor ( fig3 ). this construct was used to generate transgenic arabidopsis thaliana plants . these plants were shown to contain cbc at the same level as most of the extensin - if plants showing that the choice of signal peptide for intrinsic factor expression is not restricted to one sequence . arabidopsis thaliana was transformed with a vector construct which contained a nucleotide sequence encoding the signal peptide from the nicotiana tabacum glucan beta - 1 , 3 - glucanase fused to the nucleotide sequence for mature human intrinsic factor ( fig4 ). this construct was used to generate transgenic arabidopsis thaliana plants . these plants were shown to contain cbc at the same level as most of the extensin - if plants showing that the choice of signal peptide for intrinsic factor expression is not restricted to one sequence . as far as we have tested the recombinant human if from plants it behaves as natural human gastric if concerning its mature n - terminus , recognition by anti - if antibodies , binding of cobalamin , lack of cobinamide binding , binding to the intestinal receptor , and presence of carbohydrates . in contrast to gastric juice where if is present together with another cbp , haptocorrin , and to some extent cobalamin from the food , transgenic if plants contain no cobalamin or other cbp than if . the glycosylation of if from transgenic plants was different from human gastric if . another difference between if from human gastric mucosa and if from transgenic plants is that if from plants is at the apo - form whereas if from human beings is a mixture of the apo - and holo - form . extracts from one kilogram of transgenic arabidopsis thaliana ( tc - plants ) transformed with an extensin - transcobalamin construct ( fig5 ) contained 20 mg of recombinant human tc with cbc . western blot analysis showed a single band of approximately 45 kda which reacted with antibodies against human tc ( fig1 ). the calculated molecular weight of mature tc ( fig6 ) with 409 amino acid residues is 45536 da , showing that the observed and calculated molecular weights are similar . these results show that a plant expression system is able to produce recombinant human tc of the expected size and with cbc . the transgenic plants were analyzed as described by ( 16 ) and shown to contain no cobalamin . this shows that the recombinant human tc was at the apo - form . as for rhif , rhtc obtained from plants binds to cbl but not cobinamid ( fig1 ). n - terminal amino acid sequencing showed that the extensin signal peptide was removed from the secreted rhtc genertaing the normal mature n - terminal found in native human transcobalamin . 1 kg of the crude plant material was chopped and homogenized in 2 l of 0 . 2 m sodium phosphate buffer , ph 7 . 5 . the homogenate was centrifuged at 4000 rpm for 10 min and filtered through watman paper on a buchner funnel . the filtrate can be stored frozen at that stage . intrinsic factor was adsorbed from the solution on an affinity matrix according to a previously described method ( nexx , e ., 1975 biochim . biophys . acta 379 , 189 - 192 ). after elution from the column intrinsic factor ( saturated with cobalamin = holo - form ) was subjected to gel filtration , dialyzed against water and lyophilized . preparation of cobalamin unsaturated intrinsic factor (= apo - form ) required an additional step : dialysis against 5 m guanidine hcl for two days followed by dialysis against 0 . 2 m sodium phosphate buffer , ph 7 . 5 . 2 . rothenberg , s . p ., and quadros , e . v . 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