Methods for the production of insulin in plants

Abstract

Commercial production of human insulin can be effected via transgenic expression in plant seeds. Thus, levels of insulin accumulation exceeding 0.1% of total cellular protein can be achieved recombinantly, through expression of the insulin with a single-chain antibody as a fusion partner. Production in seeds offers flexibility in storage and shipment of insulin as a raw material, and insulin retains its activity upon extraction from stored seed. Further, the amount of biomass subjected to extraction is limited, due to the relatively low water content of plant seeds.

1 - 40 . (canceled) 
     
     
         41 . A method for the expression of insulin in plant seeds comprising:
 (a) providing a chimeric nucleic acid construct comprising in the 5′ to 3′ direction of transcription as operably linked components:
 (i) a nucleic acid sequence capable of controlling expression in plant seed cells; and 
 (ii) a nucleic acid sequence encoding an insulin polypeptide; 
   (b) introducing the chimeric nucleic acid construct into a plant cell; and   (c) growing the plant cell into a mature plant capable of setting seed wherein the seed expresses insulin.   
     
     
         42 . A method according to  claim 41  wherein the insulin polypeptide accumulates within a membrane enclosed intracellular compartment within the plant cell. 
     
     
         43 . A method according to  claim 42  wherein said membrane enclosed intracellular compartment is the endoplasmic reticulum (ER) or an ER derived storage vesicle. 
     
     
         44 . A method according to  claim 41  wherein said chimeric nucleic acid sequence additionally comprises a nucleic acid sequence encoding a polypeptide which is capable of retaining the insulin polypeptide in a membrane enclosed intracellular compartment. 
     
     
         45 . A method according to  claim 44  wherein said membrane enclosed intracellular compartment is the endoplasmic reticulum (ER) or an ER derived storage organelle. 
     
     
         46 . A method according to  claim 45  wherein said polypeptide retaining the insulin polypeptide in the ER is selected from the group consisting of KDEL, HDEL, DDEL, ADEL and SDEL. 
     
     
         47 . A method according to  claim 45  wherein said polypeptide retaining the insulin polypeptide in the ER is selected from the group consisting of SEQ ID NO:150, SEQ ID NO:151, SEQ ID NO:152, SEQ ID NO:153 and SEQ ID NO:154. 
     
     
         48 . A method according to  claim 46  wherein said insulin polypeptide additionally comprises a nucleic acid sequence encoding a signal peptide. 
     
     
         49 . A method according to  claim 48  wherein said signal peptide is a tobacco pathogenesis related protein (PR-S) signal sequence. 
     
     
         50 . A method according to  claim 48  wherein said signal sequence is SEQ.ID.NO.:161. 
     
     
         51 . A method according to  claim 45  wherein said ER-derived storage organelle is an oil body. 
     
     
         52 . A method according to  claim 45  wherein said polypeptide retaining the insulin polypeptide in an ER derived storage organelle is an oil body protein. 
     
     
         53 . A method according to  claim 42  wherein said oil body protein is selected from the group of oil body proteins consisting of oleosin, caleosin and steroleosin. 
     
     
         54 . A method according to  claim 42  wherein said oil body protein is selected from the group consisting of SEQ ID NO:156, SEQ ID NO:157 and SEQ ID NO:158, SEQ ID NO:159, and SEQ ID NO:160. 
     
     
         55 . A method according to  claim 44  wherein said chimeric nucleic acid additionally contains a nucleic acid sequence encoding a stabilizing protein fused in reading frame to the nucleic acid sequence encoding insulin. 
     
     
         56 . A method according to  claim 55  wherein said chimeric nucleic acid additionally contains a nucleic acid sequence encoding a signal peptide sequence fused in reading frame to the nucleic acid sequence encoding insulin. 
     
     
         57 . A method according to  claim 55  wherein said signal peptide is a tobacco pathogenesis related protein (PR-S) signal sequence. 
     
     
         58 . A method according to  claim 57  wherein said signal peptide is SEQ.ID.NO.:161. 
     
     
         59 . A method according to  claim 55  wherein said nucleic acid encoding said stabilizing protein permits association of the insulin polypeptide with the oil body upon harvesting and grinding of the seed. 
     
     
         60 . A method according to  claim 59  wherein said stabilizing protein encodes a single chain antibody with specificity to an oil body. 
     
     
         61 . A method according to  claim 55  wherein the nucleic acid sequence encoding a stabilizing protein fused in reading frame to the nucleic acid sequence encoding insulin is selected from the group of polypeptides consisting of a single chain antibody and cholera toxin B subunit. 
     
     
         62 . A method according to  claim 43  wherein the chimeric nucleic acid sequence is introduced in into the plant cell under nuclear genomic integration conditions. 
     
     
         63 . A method according to  claim 41  wherein said nucleic acid sequence capable of controlling expression in plant seeds is a seed-preferred promoter. 
     
     
         64 . A method according to  claim 63  wherein the seed-preferred promoter is a phaseolin promoter. 
     
     
         65 . A method according to  claim 41  wherein the nucleic acid sequence encoding insulin is selected from the group of nucleic acid sequences consisting of human insulin, porcine insulin and bovine insulin. 
     
     
         66 . A method according to  claim 41  wherein the nucleic acid encoding insulin is a mini-insulin. 
     
     
         67 . A method to  claim 41  wherein the nucleic acid sequence encoding insulin is optimized for plant codon usage. 
     
     
         68 . A method for obtaining plant seeds comprising insulin comprising:
 (a) providing a chimeric nucleic acid construct comprising in the 5′ to 3′ direction of transcription as operably linked components:
 (i) a nucleic acid sequence capable of controlling expression in plant seed cells; and 
 (ii) a nucleic acid sequence encoding an insulin polypeptide; 
   (b) introducing the chimeric nucleic acid construct into a plant cell;   (c) growing the plant cell into a mature plant capable of setting seed; and   (d) obtaining seeds from said plant wherein the seed comprises insulin.   
     
     
         69 . A method according to  claim 68  wherein at least 0.1% of the total soluble protein present in the seed is insulin. 
     
     
         70 . A plant capable of setting seed comprising a chimeric nucleic acid sequence comprising in the 5′ to 3′ direction of transcription:
 (a) a first nucleic acid sequence capable of controlling expression in a plant seed cell operatively linked to;   (b) a second nucleic acid sequence encoding an insulin polypeptide, wherein the seed contains insulin.   
     
     
         71 . A plant according to  claim 68  wherein the chimeric nucleic acid sequence is integrated in the plant's nuclear genome. 
     
     
         72 . A plant according to  claim 60  wherein the plant is an  Arabidopsis , flax or safflower plant. 
     
     
         73 . A plant seed comprising a chimeric nucleic acid sequence comprising in the 5′ to 3′ direction of transcription:
 (a) a first nucleic acid sequence capable of controlling expression in a plant seed cell operatively linked to;   (b) a second nucleic acid sequence encoding an insulin polypeptide.   
     
     
         74 . Plant seed according to  claim 73  wherein at least 0.1% of the total soluble protein present in the seed is insulin. 
     
     
         75 . A nucleic acid sequence encoding insulin linked to nucleic acid sequence comprising a promoter capable of controlling expression in a plant seed cell. 
     
     
         76 . A nucleic acid sequence according to  claim 75  wherein said promoter is a seed preferred promoter. 
     
     
         77 . A nucleic acid sequence according to  claim 76  wherein said seed preferred promoter is a phaseolin promoter. 
     
     
         78 . A nucleic acid sequence according to  claim 75  wherein said nucleic acid sequence additionally contains a sequence capable of retaining the insulin polypeptide in a membrane enclosed intracellular compartment. 
     
     
         79 . A nucleic acid sequence according to  claim 75  wherein said nucleic acid sequence additionally contains a sequence capable of retaining the insulin polypeptide in the ER or an ER derived storage organelle. 
     
     
         80 . Use of a plant seed prepared according to claim  40  to obtain substantially pure insulin.