Process for producing plant storage organ with high production of recombinant protein and novel recombinant protein

Abstract

The present invention provides a method for highly producing a recombinant protein in a plant storage organ and a GLP-1 derivative. The plant storage organ in which the recombinant protein is highly produced is obtained by transformation with the use of a vector which comprises a recombinant protein gene, a cytokinin-related gene, a drug-resistant gene and a removable DNA element, in which the cytokinin-related gene and the drug-resistant gene exist in the positions so that they can behave together with the DNA element, while the recombinant protein to be expressed in the plant storage organ exists in the position so that it would not behave together with the DNA element. The GLP-1 is produced by using the method, and a derivative having been stabilized against enzymatic digestion is further provided.

1 - 18 . (canceled) 
     
     
         19 . A method for producing a plant storage organ in which a GLP-1 derivative recombinant protein is highly produced, wherein a gene encoding the GLP-1 derivative in which glutamine is substituted at the 26th position and asparagine or asparatic acid is substituted at the 34th position in a peptide comprising GLP-1 (7-36) or its amino acid sequence in which one or a few amino acids are deleted, substituted and/or added, and having a GLP-1 activity is used as a recombinant protein gene to be expressed in a plant storage organ, and which method comprises the following steps (A), (B), and (C):
 (A) a step of constructing a vector which comprises a recombinant protein gene to be expressed in a plant storage organ, a cytokinin-related gene, a drug-resistant gene, and a removable DNA element, wherein the cytokinin-related gene and the drug-resistant gene exist in the positions so that they can behave together with the removable DNA element, while the recombinant protein gene to be expressed in the plant storage organ exists in the position so that it would not behave together with the removable DNA element, and introducing the vector into cells,   (B) a step of redifferentiating transformant by culturing the plant cell into which the vector is introduced by said step (A) in a drug-supplemented medium and a drug-free medium, and   (C) a step of obtaining the plant storage organ from the transformant redifferentiated in said step (B).   
     
     
         20 . The method for producing a plant storage organ in which a GLP-1 derivative recombinant protein is highly produced according to  claim 19 , wherein the gene encoding GLP-1 derivative is a gene encoding GLP-1 derivative in which serine or glycine is further substituted at the 8th position in the amino acid sequence. 
     
     
         21 . The method for producing a plant storage organ in which a GLP-1 derivative recombinant protein is highly produced according to  claim 20 , wherein the gene encoding GLP-1 derivative is a gene shown in SEQ ID No:1 of the sequence listing. 
     
     
         22 . The method for producing a plant storage organ in which a GLP-1 derivative recombinant protein is highly produced according to  claim 19 , wherein the GLP-1 derivative consists of the amino acid sequence shown in SEQ ID No: 2, 5 or 6 of the sequence listing. 
     
     
         23 . The method for producing a plant storage organ in which a GLP-1 derivative recombinant protein is highly produced according to  claim 19 , comprising culturing the plant cell into which the vector is introduced in a plant hormone-free medium and drug-free medium after culturing it in a plant hormone-supplemented medium and drug-supplemented medium, during the step of redifferentiating transformant from the plant cell into which the vector has been introduced. 
     
     
         24 . The method for producing a plant storage organ in which a GLP-1 derivative recombinant protein is highly produced according to  claim 19 , wherein the gene encoding GLP-1 derivative is under control of a promoter specific to the plant storage organ. 
     
     
         25 . The method for producing a plant storage organ in which a GLP-1 derivative recombinant protein is highly produced according to  claim 19 , wherein the gene encoding GLP-1 derivative is inserted into or is substituted for the site encoding protein variable region, in the gene encoding the protein originally expressed in the plant storage organ. 
     
     
         26 . The method for producing a plant storage organ in which a GLP-1 derivative recombinant protein is highly produced according to  claim 25 , wherein a nucleotide sequence which encodes an amino acid sequence for enzyme cleavage to cleave and separate the GLP-1 derivative from the protein originally expressed in the plant storage organ is placed into the boundary between the gene encoding GLP-1 derivative and the gene encoding the protein originally expressed in the plant storage organ. 
     
     
         27 . The method for producing a plant storage organ in which a GLP-1 derivative recombinant protein is highly produced according to  claim 19 , wherein the plant storage organ is a seed. 
     
     
         28 . The method for producing a plant storage organ in which a GLP-1 derivative recombinant protein is highly produced according to  claim 27 , wherein the gene encoding the protein originally expressed in the plant storage organ to be inserted into or to be substituted for the protein variable region is a seed storage protein gene. 
     
     
         29 . The method for producing a plant storage organ in which a GLP-1 derivative recombinant protein is highly produced according to  claim 19 , wherein the cytokinin-related gene is a cytokinin-synthesis gene. 
     
     
         30 . The method for producing a plant storage organ in which a GLP-1 derivative recombinant protein is highly produced according to  claim 29 , wherein the cytokinin-synthesis gene is an isopentenyl transferase gene. 
     
     
         31 . The method for producing a plant storage organ in which a GLP-1 derivative recombinant protein is highly produced according to  claim 19 , wherein the drug-resistant gene is a hygromycin-resistant gene. 
     
     
         32 . The method for producing a plant storage organ in which a GLP-1 derivative recombinant protein is highly produced according to  claim 19 , wherein the removable DNA element is derived from a site-specific recombination system or a transposon. 
     
     
         33 . The method for producing a plant storage organ in which a GLP-1 derivative recombinant protein is highly produced according to  claim 19 , wherein the plant is monocotyledon. 
     
     
         34 . The method for producing a plant storage organ in which a GLP-1 derivative recombinant protein is highly produced according to  claim 33 , wherein the monocotyledon is  Oryza sativa.    
     
     
         35 . A plant storage organ in which a GLP-1 derivative recombinant protein is highly produced by the method for producing according to  claim 19 , or a transformed plant to produce the plant storage organ. 
     
     
         36 . A recombinant vector for introducing a gene into a plant to use in the method for producing a plant storage organ in which a GLP-1 derivative recombinant protein is highly produced according to  claim 19 , wherein a gene encoding the GLP-1 derivative in which glutamine is substituted at the 26th position and asparagine or asparatic acid is substituted at the 34th position in a peptide comprising GLP-1 (7-36) or its amino acid sequence in which one or a few amino acids are deleted, substituted and/or added, and having a GLP-1 activity is used as a recombinant protein gene to be expressed in a plant storage organ; and which recombinant vector comprises a gene encoding the GLP-1 derivative; a cytokinin-related gene, a drug-resistant gene and a removable DNA element; and which gene has been introduced into a vector wherein the cytokinin-related gene and the drug-resistant gene exist in the positions so that they can behave together with the removable DNA element, while the recombinant protein gene to be expressed in the plant storage organ exists in the position so that it would not behave together with the removable DNA element. 
     
     
         37 . The vector for gene introduction into a plant according to  claim 36 , wherein the gene encoding GLP-1 derivative is a gene encoding a peptide in which serine or glycine is further substituted at the 8th position in the amino acid sequence. 
     
     
         38 . The vector for gene introduction into a plant according to  claim 37 , wherein the gene encoding GLP-1 derivative is a gene shown in SEQ ID No:1 of the sequence listing.