Transgenic plants expressing cobalamin binding proteins

Abstract

The present invention relates to the use of transgenic plants for the expression of vitamin B12 (cobalamin) 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.

1 . A transgenic plant that expresses intrinsic factor or a protein with at least 60% sequence identity to intrinsic factor or a functional fragment thereof capable of binding cobalamin and/or an analog thereof.  
     
     
         2 . The transgenic plant according to  claim 1 , wherein said protein is in the apoform.  
     
     
         3 . The transgenic plant according to  claim 1 , wherein the plant is “Generally Recognized A Safe” (GRAS).  
     
     
         4 . The transgenic plant according to  claim 1 , wherein the plant is  Arabidopsis thaliana.    
     
     
         5 . Propagating material derived from a transgenic plant as defined in  claim 1 .  
     
     
         6 . The propagating material according to  claim 5 , which comprises seeds.  
     
     
         7 . Intrinsic factor or a protein with at least 60% sequence identity to intrinsic factor or a functional fragment thereof that is expressed in a transgenic plant and is capable of binding cobalamin and/or an analog thereof.  
     
     
         8 . Intrinsic factor or a protein with at least 60% sequence identity to intrinsic factor or a functional fragment thereof according to  claim 7 , which is isolated and/or purified from the transgenic plant.  
     
     
         9 . Intrinsic factor or a protein with at least 60% sequence identity to intrinsic factor or a functional fragment thereof according to  claim 7 , said protein being in the apoform.  
     
     
         10 . A method of isolating and/or purifying intrinsic factor or a protein with at least 60% sequence identity to intrinsic factor or a functional fragment thereof as defined in  claim 7 , which includes the steps of: 
 homogenizing the transgenic plant.    
     
     
         11 . The method according to  claim 10 , which comprises the following steps: 
 filtration of the supernatant formed by centrifugation of the homogenate,    affinity column chromatography using cobalamin,    elution of the cobalamin binding protein attached to the cobalamin,    gel filtration; and    dialysis against water;    and optionally further comprising the following steps to produce the apoform of the cobalamin:    dialysis against 5M guanidine HCl; and    dialysis against 0.2M sodium phosphate.    
     
     
         12 . A composition comprising a cobalamin binding protein or a functional fragment thereof as defined in  claim 7 .  
     
     
         13 . The composition according to  claim 12 , which is a pharmaceutical composition and optionally further comprises one or more pharmaceutically acceptable diluents, excipients and/or carriers.  
     
     
         14 . The composition according to  claim 12 , further comprising a second component selected from cobalamin, cobalamin analog or cobalamin containing a tracer molecule.  
     
     
         15 . The composition according to  claim 12  which is for oral use.  
     
     
         16 . A foodstuff comprising transgenic plants as defined in  claim 1  or a plant material derived from said plant.  
     
     
         17 . The use of a protein as defined in  claim 7  in diagnostic tests.  
     
     
         18 . A method for quantifying cobalamin absorption from the intestine by means of a Schilling test utilizing a protein as defined in  claim 7 , wherein the protein is intrinsic factor or a functional fragment thereof.  
     
     
         19 . A method for quantifying a cobalamin binding protein utilizing a protein as defined in  claim 7 .  
     
     
         20 . A method for quantifying a cobalamin binding protein utilizing a protein as defined in  claim 7 , or antibodies against said protein.  
     
     
         21 . A method for quantifying receptors for a cobalamin binding protein utilizing a protein as defined in  claim 7 .  
     
     
         22 . The method according to  claim 21  further utilizing labelled cobalamin.  
     
     
         23 . The method according to  claim 22 , wherein the cobalamin is radioactively labelled.  
     
     
         24 . A nucleic acid construct comprising nucleic acid coding for intrinsic factor or a protein with at least 60% sequence identity to intrinsic factor or a functional fragment thereof, operably linked to one or more regulatory sequences capable of directing expression in a plant.  
     
     
         25 . The nucleic acid construct according to  claim 24 , wherein the nucleic acid is DNA.  
     
     
         26 . A nucleic acid according to  claim 24 , wherein the regulatory sequences are selected from any one of  Arabidopsis thaliana  extensin signal peptide,  Phalsoelus vulgaris  chitinase signal peptide,  Nicotiana tabacum  glucan beta-1,3-glucanase and/or 35S promoter.  
     
     
         27 . A vector comprising a nucleic acid construct as defined in  claim 24 .  
     
     
         28 . A plant cell comprising a vector as claimed in  claim 27 .  
     
     
         29 . A whole plant or part thereof, comprising a plant cell as defined in  claim 28 .  
     
     
         30 . A method for treating cobalamin deficiency comprising administering a protein or a functional fragment thereof as defined in  claim 7  to a subject in need thereof.  
     
     
         31 . The method according to  claim 30 , wherein the cobalamin deficiency is caused by low levels of at least one of intrinsic factor, transcobalamin and haptocorrin.  
     
     
         32 . Cancel  
     
     
         33 . Cancel