Claims for Patent: 5,650,554

Summary for Patent: 5,650,554

Title:	Oil-body proteins as carriers of high-value peptides in plants
Abstract:	The present invention relates to the use of a class of genes called oil body protein genes that have unique features. The discovery of these features allowed the invention of methods for the production of recombinant proteins wherein a protein of interest can be easily separated from other host cell components. The invention is further exemplified by methods for exploitation of the unique characteristics of the oil body proteins and oil body genes for expression of polypeptides of interest in many organisms, particularly plant seeds. Said polypeptides may include but are not limited to: seed storage proteins, enzymes, bioactive peptides, antibodies and the like. The invention can also be modified to recover recombinant polypeptides fused to oleosins from non-plant host cells. Additionally the invention provides a method of using recombinant proteins associated with seed oil bodies released during seed germination for expression of polypeptides that afford protection to seedlings from pathogens. Finally, the persistent association of oil body proteins with the oil body can be further utilized to develop a biological means to create novel immobilized enzymes useful for bioconversion of substrates.
Inventor(s):	Moloney; Maurice (Calgary, CA)
Assignee:	SemBioSys Genetics Inc. (Calgary, CA)
Application Number:	08/366,783
Patent Claims:	1. A method for the expression of a recombinant polypeptide by a plant or bacterial host cell said method comprising: a) introducing into a plant or bacterial host cell, a 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 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; and b) growing said host cell to produce the recombinant fusion polypeptide. 2. The method according to claim 1 further including separating the recombinant fusion polypeptide from cellular host cell components by selective partitioning into a lipid phase. 3. The method according to claim 1 further including separating the recombinant fusion polypeptide from cellular host components by selective partitioning into a lipid phase comprising oil bodies. 4. The method according to claim 3 wherein said recombinant fusion polypeptide is separated by addition of oil body components and rcconstitution of the oil bodies. 5. The method according to claim 2 further comprising releasing the recombinant polypeptide from the recombinant fusion polypeptide associated with the lipid phase, said method comprising: c) including in said second DNA sequence (2) between said DNA sequence (i) encoding the oleosin protein and the DNA sequence (ii) encoding the recombinant polypeptide, a linker DNA sequence (iii) encoding an amino acid sequence that is specifically cleavable by enzymatic or chemical means; and d) contacting the lipid phase with said enzymatic or chemical means such that said recombinant polypeptide is released from the recombinant fusion polypeptide. 6. The method according to claim 5 wherein said amino acid sequence encoded by said linker DNA sequence is cleavable by enzymatic means. 7. The method according to claim 6 wherein said linker DNA sequence encodes an amino acid sequence that is recognizable by the proteolytic action of an enzyme selected from the group consisting of thrombin, factor Xa, collagenase and chymosin. 8. The method according to claim 6 wherein said enzymatic means comprises an enzyme that is immobilized. 9. The method according to claim 8 wherein said enzyme is immobilized by attachment to an oleosin protein that is associated with an oil body. 10. The method according to claim 1 wherein said recombinant polypeptide is an enzyme. 11. The method according to claim 10 wherein said recombinant polypeptide is an enzyme that retains its enzymatic properties while part of the recombinant fusion polypeptide associated with the oil body. 12. 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. 13. 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. 14. 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. 15. The method according to claim 14 wherein said enzyme is activated by lowering the pH of the oil body environment. 16. 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. 17. The method according to claim 1 wherein said recombinant polypeptide is an interleukin. 18. The method according to claim 1 wherein said recombinant polypeptide is a thrombin inhibitor. 19. The method according to claim 1 wherein said recombinant polypeptide is hirudin. 20. The method according to claim 1 wherein said host cell is a plant cell. 21. The method according to claim 20 wherein said plant is dicotyledonous. 22. The method according to claim 20 wherein said plant is from the family Brassicaceae. 23. The method according to claim 1 wherein said host cell is a bacterial cell. 24. The method according to claim 1 wherein in said second DNA sequence, the DNA sequence (i) is an oleosin gene derived from a plant from the family Brassicaceae. 25. The method accordingly to claim 1 wherein in said second DNA sequence, the DNA sequence (i) is an oleosin gene derived from Arabidopsis thaliana. 26. The method according to claim 25 wherein said first DNA sequence (1) is derived from an oleosin gene from the plant Arabidopsis thaliana. 27. The method according to claim 25 wherein said DNA sequence (i) has the sequence as shown in SEQ ID NO. 1. 28. The method according to claim 25 wherein said DNA sequence (i) encodes a polypeptide having the amino acid sequence as shown in SEQ ID NO. 5. 29. 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. 30. The chimeric DNA sequence according to claim 29 wherein said DNA sequence (ii) encodes an enzyme. 31. The chimeric DNA sequence according to claim 29 further including (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. 32. The chimeric DNA sequence according to claim 31 wherein said linker DNA sequence (iii) encodes a cleavage site for an enzyme selected from the group consisting of thrombin, factor Xa, collagenase and chymosin. 33. The chimeric DNA sequence according to claim 29 wherein said DNA sequence (ii) encodes an interleukin. 34. The chimeric DNA sequence according to claim 29 wherein said DNA sequence (ii) encodes a thrombin inhibitor. 35. The chimeric DNA sequence according to claim 29 wherein said DNA sequence (ii) encodes hirudin. 36. The chimeric DNA sequence according to claim 29 wherein said DNA sequence (i) is an oleosin gene derived from a plant from the family Brassicaceae. 37. The chimeric DNA sequence according to claim 29 wherein said DNA sequence (i) is an oleosin gene derived from Arabidopsis thaliana. 38. The chimeric DNA sequence according to claim 29 wherein said first DNA sequence (1) is derived from an oleosin gene from the plant Arabidopsis thaliana. 39. The chimeric DNA sequence according to claim 29 wherein said DNA sequence (i) has the sequence as shown in SEQ ID NO. 1. 40. The chimeric DNA sequence according to claim 29 wherein said DNA sequence (i) encodes a polypeptide having the amino acid sequence as shown in SEQ ID NO. 5. 41. An expression cassette comprising a chimeric DNA sequence according to claim 29. 42. A plant transformed with a chimeric DNA sequence according to claim 29. 43. A plant cell culture containing a chimeric DNA sequence according to claim 29. 44. A plant seed containing a chimeric DNA sequence according to claim 29.

Details for Patent 5,650,554

Applicant	Tradename	Biologic Ingredient	Dosage Form	BLA	Approval Date	Patent No.	Expiredate
Smith & Nephew, Inc.	SANTYL	collagenase	Ointment	101995	06/04/1965		2014-07-22