Oil-body proteins as carriers of high-value peptides in plants

A method for the production and release of a recombinant polypeptide from a recombinant fusion polypeptide associated with a plant oil body fraction during seed germination and plant seedling growth, said method comprising:

• a) introducing into a plant cell a first chimeric DNA sequence comprising:
• 1) a first DNA sequence capable of regulating the transcription in said plant cell of
• 2) a second DNA sequence wherein said DNA second sequence encodes a recombinant fusion polypeptide and comprises (i) a DNA sequence encoding a sufficient portion of an oleosin protein to provide targeting of the recombinant fusion polypeptide to an oil body, linked in reading frame to (ii) a DNA sequence encoding a recombinant polypeptide and (iii) a linker DNA sequence encoding an amino acid sequence that is specifically cleavable by enzymatic means wherein said linker DNA sequence (iii) is located between said DNA sequence (i) encoding the oleosin protein and said DNA sequence (ii) encoding the recombinant polypeptide;
• and
• 3) a third DNA sequence encoding a termination region;
• b) sequentially or concomitantly introducing into the genome of said plant a second chimeric DNA sequence comprising:
• 1) a first DNA sequence capable of regulating the transcription specifically during seed germination and seed growth of
• 2) a second DNA sequence encoding a specific enzyme that is capable of cleaving the linker DNA sequence of said first chimeric DNA sequence;
• and
• 3) a third DNA sequence encoding a termination region;
• c) regenerating a plant from said plant cell and growing said plant to produce seed whereby said recombinant fusion polypeptide is expressed and associated with oil bodies and
• d) allowing said seed to germinate wherein said enzyme encoded by said second chimeric DNA sequence is expressed and cleaves the recombinant polypeptide from the recombinant fusion polypeptide associated with the oil bodies during seed germination and early seedling growth.

A method for producing an altered seed meal by producing a recombinant polypeptide in association with a plant seed oil body fraction, said method comprising:

• a) introducing into a plant cell a chimeric DNA sequence comprising:
• 1) a first DNA sequence capable of regulating the transcription in said plant cell of
• 2) a second DNA sequence wherein said second sequence encodes a recombinant fusion polypeptide and comprises (i) a DNA sequence encoding a sufficient portion of an oleosin protein to provide targeting of the recombinant fusion polypeptide to an oil body, linked in reading frame to (ii) a DNA sequence encoding a recombinant polypeptide and
• 3) a third DNA sequence encoding a termination region;
• b) regenerating a plant from said plant cell and growing said plant to produce seed whereby said recombinant polypeptide is expressed and associated with oil bodies;
• and
• c) crushing said seed and preparing an altered seed meal.

A method of preparing an enzyme in a plant or bacterial host cell in association with an oil body and releasing said enzyme from the oil body, said method comprising:

• a) transforming a plant or bacterial host cell with a chimeric DNA sequence comprising:
• 1) a first DNA sequence capable of regulating the transcription of
• 2) a second DNA sequence, wherein said second sequence encodes a recombinant fusion polypeptide and comprises (i) a DNA sequence encoding a sufficient portion of an oleosin protein to provide targeting of the recombinant fusion polypeptide to an oil body;
• (ii) a DNA sequence encoding an enzyme and (iii) a linker DNA sequence located between said DNA sequence (i) encoding the oleosin protein and said DNA sequence (ii) encoding the enzyme wherein said DNA sequence (iii) encodes an amino acid sequence that is cleavable by the enzyme encoded by the DNA sequence (ii);
• and
• 3) a third DNA sequence encoding a termination region functional in said host cell
• b) growing the host cell to produce the recombinant fusion polypeptide under conditions such that enzyme is not active;
• c) recovering the oil bodies containing the recombinant fusion polypeptide;
• and
• d) altering the environment of the oil bodies such that the enzyme is activated and cleaves itself from the recombinant fusion polypeptide.

A method for the expression of a recombinant polypeptide by a plant or bacterial host cell in association with an oil body and separating said recombinant polypeptide from the oil body, said method comprising:

• a) transforming a first plant or bacterial host cell with a first chimeric DNA sequence comprising:
• 1) a first DNA sequence capable of regulating the transcription in said host cell of
• 2) a second DNA sequence, wherein said second sequence encodes a first recombinant fusion polypeptide and comprises (i) a DNA sequence encoding a sufficient portion of an oleosin protein to provide targeting of the recombinant fusion polypeptide to a lipid phase, linked in reading frame to (ii) a DNA sequence encoding said recombinant polypeptide;
• and (iii) a linker DNA sequence encoding an amino acid sequence that is specifically cleavable by enzymatic means wherein said linker DNA sequence (iii) is located between said (i) DNA sequence encoding the oleosin protein and said (ii) DNA sequence encoding the recombinant polypeptide;
• and
• 3) a third DNA sequence encoding a termination region functional in the host cell;
• and
• b) transforming a second plant or bacterial host cell with a second chimeric DNA sequence comprising:
• 1) a first DNA sequence capable of regulating the transcription specifically during seed germination and seed growth of
• 2) a second DNA sequence wherein said second sequence encodes a second recombinant fusion polpeptide and comprises (i) a DNA sequence encoding a sufficient portion of an oleosin protein to provide targeting of the second recombinant fusion polypeptide to a lipid phase linked in reading frame to do a DNA sequence, encoding a specific enzyme that is capable of cleaving the linker DNA sequence of said first chimeric DNA sequence;
• and
• 3) a third DNA sequence encoding a termination region;
• c) growing said first host cell under conditions such that the first recombinant fusion polypeptide is expressed and associated with the oil bodies to produce a first oil body fraction containing the first recombinant fusion polypeptide;
• d) growing said second host cell under conditions such that the second recombinant fusion polypeptide is expressed and associated with the oil bodies to product a second oil body fraction containing the second recombinant fusion polypeptide;
• e) contacting the first oil body fraction of step (c) with the second oil body fraction of step (d) under conditions such that the enzyme portion of the second recombinant fusion polypeptide cleaves the first recombinant polypeptide from the first recombinant fusion polypeptide.

A chimeric DNA sequence, capable of being expressed in association with an oil body of a plant or bacterial host cell, comprising:

• 1) a first DNA sequence capable of regulating the transcription in said plant or bacterial host cell of
• 2) a second DNA sequence, wherein said second sequence encodes a recombinant fusion polypeptide and comprises (i) a DNA sequence encoding a sufficient portion of an oleosin protein to provide targeting of the recombinant fusion polypeptide to a lipid phase, linked in reading frame to (ii) a DNA sequence encoding said recombinant polypeptide;
• and
• 3) a third DNA sequence encoding a termination region functional in the host cell.