Commercial production of chymosin in plants

Abstract

A method for the commercial production of chymosin which involves the recombinant expression of chymosin in plant seeds, that enables chymosin to accumulate to at least 0.5% (w/w) of total seed protein is described. An improved method for the isolation of chymosin from the seed is also described.

We claim: 
     
         1 . A method for the production of chymosin in a plant seed comprising:
 a) introducing into a plant cell a chimeric nucleic acid sequence molecule comprising in the 5′ to 3′ direction of transcription:
 1) a first nucleic acid sequence capable of regulating transcription in said plant cell operatively linked to; 
 2) a second nucleic acid sequence encoding a chymosin polypeptide operatively linked to; 
 3) a third nucleic acid sequence capable of terminating transcription in said plant cell; 
   b) growing said plant cell into a mature plant capable of setting seed; and   c) obtaining seed from the mature plant wherein said seed contains chymosin.   
     
     
         2 . The method according to  claim 1  wherein said first nucleic sequence capable of regulating transcription in said plant cell is a seed-specific promoter. 
     
     
         3 . The method according to  claim 3  wherein said seed-specific promoter is a phaseolin promoter. 
     
     
         4 . A method according to  claim 1  wherein at least 0.5% (w/w) of the total seed protein is chymosin. 
     
     
         5 . The method according to  claim 1  wherein the second nucleic acid sequence encoding a chymosin polypeptide comprises a nucleic acid sequence encoding a chymosin pro-peptide, a nucleic acid sequence encoding a chymosin pre-peptide or a nucleic acid sequence encoding chymosin pre-pro-peptide. 
     
     
         6 . The method according to  claim 5  wherein the second nucleic acid sequence encoding a chymosin polypeptide further comprises a nucleic acid sequence encoding a plant signal sequence. 
     
     
         7 . The method according to  claim 1  wherein the second nucleic acid sequence encoding a chymosin polypeptide further comprises a nucleic acid sequence encoding a plant signal sequence. 
     
     
         8 . The method according to  claim 7  wherein the plant signal sequence is a tobacco PR-S sequence. 
     
     
         9 . The method according to  claim 8  wherein the nucleic acid sequence encoding chymosin linked to a PR-S signal sequence comprises a nucleic acid sequence as in SEQ.ID.NO.:1. 
     
     
         10 . The method according to  claim 1  wherein said third nucleic acid sequence is a phaseolin terminator. 
     
     
         11 . The method according to  claim 1  wherein the chymosin is a mammalian chymosin obtainable from a bovine, sheep or goat source. 
     
     
         12 . The method according to  claim 6  wherein codon usage for said nucleic acid sequence encoding chymosin, chymosin pro-peptide, chymosin pre-peptide and chymosin pre-pro-peptide has been optimized for use in plants. 
     
     
         13 . The method according to  claim 1  wherein said plant is selected from the group of plants consisting of soybean ( Glycine max ), rapeseed ( Brassica napus, Brassica campestris ), sunflower ( Helianthus annuus ), cotton ( Gossypium hirsutum ), corn ( Zea mays ), tobacco ( Nicotiana tobacum ), alfalfa ( Medicago sativa ), wheat ( Triticum  sp.), barley ( Hordeum vulgare ), oats ( Avena sativa  L.), sorghum ( Sorghum bicolor ),  Arabidopsis thaliana , potato ( Solanum  sp.), flax/linseed ( Linum usitatissimum ), safflower ( Carthamus tinctorius ), oil palm ( Eleais guineeis ), groundnut ( Arachis hypogaea ), Brazil nut ( Bertholletia excelsa ) coconut ( Cocus nucifera ), castor ( Ricinus communis ), coriander ( Coriandrum sativum ), squash ( Cucurbita maxima ), jojoba ( Simmondsia chinensis ) and rice ( Oryza sativa ). 
     
     
         14 . The method according to  claim 1  wherein at least 1% (w/w) of said total seed protein is chymosin. 
     
     
         15 . The method according to  claim 1  wherein at least 2% (w/w) of said total seed protein is chymosin. 
     
     
         16 . The method according to  claim 1  wherein at least 4% (w/w) of said total seed protein is chymosin. 
     
     
         17 . A method for the production of plant seeds containing at least 0.5% (w/w) chymosin in the total seed protein comprising:
 (a) introducing into each of at least two plant cells a chimeric nucleic acid sequence molecule comprising in the 5′ to 3′ direction of transcription:
 1) a first nucleic acid sequence capable of regulating transcription in said plant cell operatively linked to; 
 2) a second nucleic acid sequence encoding a chymosin polypeptide operatively linked to; 
 3) a third nucleic acid sequence capable of terminating transcription in said plant cell; 
   (b) growing each plant cell into a mature plant capable of setting seed;   (c) obtaining seed from each mature plant;   (d) detecting the levels of chymosin in the seed of each plant obtained in step (c) or in the seed of a plant generated from the seed of a plant obtained in step (c); and   (e) selecting plants that contain at least 0.5% (w/w) chymosin in the total seed protein.   
     
     
         18 . A method according to  claim 1  further comprising (d) isolating said chymosin from said seed. 
     
     
         19 . A method according to  claim 18  wherein (d) isolating said chymosin from said seed comprises:
 (i) crushing the plant seed to obtain crushed plant seed; 
 (ii) contacting the crushed plant seed or a fraction thereof with a protein binding resin; and 
 (iii) recovering chymosin from the protein binding resin. 
 
     
     
         20 . A method according to  claim 18  wherein (d) isolating said chymosin from said seed comprises:
 (i) crushing of the plant seed to obtain crushed plant seed; 
 (ii) fractionating the crushed plant seed into an oil fraction, aqueous fraction and a fraction comprising insoluble material; 
 (iii) contacting the aqueous fraction with a protein binding resin; and 
 (iv) recovering the chymosin from the protein binding resin. 
 
     
     
         21 . A method according to  claim 19  wherein said protein binding resin is a hydrophobic interaction resin. 
     
     
         22 . A method according to  claim 20  wherein said protein binding resin is a hydrophobic interaction resin. 
     
     
         23 . A method according to  claim 22  further comprising using an ion exchange resin to further purify the chymosin. 
     
     
         24 . Plant seed comprising at least 0.5% (w/w) heterologously expressed chymosin. 
     
     
         25 . Plant seed prepared according to the method of  claim 1 . 
     
     
         26 . Plant seed prepared according to the method of  claim 17 . 
     
     
         27 . A plant capable of setting seed comprising at least 0.5% (w/w) of heterologously expressed chymosin. 
     
     
         28 . A plant capable of setting seed prepared according to the method of  claim 1 .