Preparation of heterologous proteins on oil bodies

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

• a) introducing into a plant cell a chimeric nucleic acid sequence comprising:
• 1) a first nucleic acid sequence capable of regulating the transcription in said plant cell of
• 2) a second nucleic acid sequence wherein said second sequence encodes a fusion polypeptide and comprises (i) a nucleic acid sequence encoding a sufficient portion of an oil body protein gene to provide targeting of the fusion polypeptide to an oil body, linked in reading frame to (ii) a nucleic acid sequence encoding the heterologous polypeptide and
• 3) a third nucleic add sequence encoding a termination region;
• b) regenerating a plant from said plant cell and growing said plant to produce seed whereby said heterologous 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 host cell in association with an oil body and releasing said enzyme from the oil body, said method comprising:

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

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

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

A method for the expression of a heterologous polypeptide by a host cell said method comprising:

• a) generating by homologous recombination into a host cell a chimeric nucleic acid sequence comprising:
• 1) a first nucleic acid sequence capable of regulating transcription in said host cell
• 2) a second nucleic acid sequence, wherein said second sequence encodes a fusion polypeptide and comprises (i) a nucleic acid sequence encoding a sufficient amount of an oil body protein gene to provide targeting of the fusion polypeptide to a lipid phase, linked in reading frame to (ii) a nucleic acid sequence encosing the heterologous polypeptide;
• and
• 3) a third nucleic acid sequence encoding a termination region functional in the host cell;
• and
• b) growing said hot cell to produce the heterologous polypeptide.

A chimeric nucleic acid sequence, capable of being expressed in association with an oil body of a host cell comprising:

• 1) a first nucleic acid sequence capable of regulating the transcription in said host cell of
• 2) a second nucleic acid sequence, wherein said second sequence encodes a fusion polypeptide and comprises (i) a nucleic acid sequence encoding a sufficient portion of an oil body protein gene to provide targeting of the fusion polypeptide to a lipid phase linked in reading frame to (ii) a nucleic acid sequence encoding the heterologous polypeptide;
• and
• 3) a third nucleic acid sequence encoding a termination region functional in the host cell.