Methods for the production of multimeric protein complexes, and related compositions

• US 20030167524A1
• Filed: 12/19/2001
• Published: 09/04/2003
• Est. Priority Date: 12/19/2000
• Status: Abandoned Application

• Current Assignee: Syngenta Participations AG (China National Chemical Corporation), Sembiosys Genetics Inc. (Corteva, Inc.)
• Original Assignee: Syngenta Participations AG (China National Chemical Corporation)
• Inventors: Zaplachinski, Steven, Rooijen, Gijs Van, Heifetz, Peter-Bernard, Dalmia, Bipin Kumar, Briggs, Steven, Val, Greg Del
• Application Number:
  US10/032,201
• Publication Number:
  US 20030167524A1
• Time in Patent Office: Days
• US Class Current: 800/281
• CPC Class Codes: C12N 15/8257 for the production of primary gene products, e.g. pharmaceutical products, interferon
• CPC Class Codes: C12N 15/8258 for the production of oral vaccines (antigens) or immunoglobulins

Abstract

Improved methods for the production of multimeric-protein-complexes, such as redox proteins and immunoglobulins, in association with oil bodies are described. The redox protein is enzymatically active when prepared in association with the oil bodies. Also provided are related nucleic acids, proteins, cells, plants, and compositions.

Claims

1. A method of producing an oil body associated with a recombinant multimeric-protein-complex, said method comprising:

(a) producing in a cell comprising oil bodies, a first recombinant polypeptide and a second recombinant polypeptide wherein said first recombinant polypeptide is capable of associating with said second recombinant polypeptide to form said multimeric-protein-complex; and

(b) associating said multimeric-protein-complex with an oil body through an oil-body-targeting-protein capable of associating with said oil body and said first recombinant polypeptide,

wherein said first recombinant polypeptide is a thioredoxin and said second recombinant polypeptide is a thioredoxin-reductase.

2. A method of expressing a recombinant multimeric-protein-complex comprising a first and second recombinant polypeptide in a cell, said method comprising:

(a) introducing into a cell a first chimeric nucleic acid sequence comprising;

(i) a first nucleic acid sequence capable of regulating transcription in said cell operatively linked to;

(ii) a second nucleic acid sequence encoding a first recombinant polypeptide;

(b) introducing into said cell a second chimeric nucleic acid sequence comprising;

(i) a third nucleic acid sequence capable of regulating transcription in said cell operatively linked to;

(ii) a fourth nucleic acid sequence encoding a second recombinant polypeptide;

(c) growing said cell under conditions to permit expression of said first and second recombinant polypeptide in a progeny cell comprising oil bodies wherein said first recombinant polypeptide and said second recombinant polypeptide are capable of forming a multimeric-protein-complex; and

(d) associating said first recombinant polypeptide with an oil body through an oil-body-targeting-protein capable of associating with said oil body and said first recombinant polypeptide,

wherein said first recombinant polypeptide is a thioredoxin and said second recombinant polypeptide is a thioredoxin-reductase.

3. A method of producing in a plant a recombinant multimeric-protein-complex, said method comprising:

(a) preparing a first plant comprising cells, said cells comprising oil bodies and a first recombinant polypeptide wherein said first recombinant polypeptide is capable of associating with said oil bodies through an oil-body-targeting-protein;

(b) preparing a second plant comprising cells, said cells comprising oil bodies and a second recombinant polypeptide; and

(c) sexually crossing said first plant with said second plant to produce a progeny plant comprising cells, said cells comprising oil bodies, wherein said oil bodies are capable of associating with said first recombinant polypeptide, and said first recombinant recombinant polypeptide is capable of associating with said second recombinant polypeptide to form said recombinant multimeric-protein-complex,

wherein said first recombinant polypeptide is a thioredoxin and said second recombinant polypeptide is a thioredoxin-reductase.

13. A method of treating or protecting a target against oxidative stress, comprising the steps of:

providing the recombinant fusion polypeptide of claim 3;

and

contacting the recombinant fusion polypeptide with a target, wherein the target is susceptible to oxidative stress, thereby treating or protecting against the stress.

4. A chimeric nucleic acid sequence encoding a multimeric-fusion-protein, said nucleic acid comprising:

(a) a first nucleic acid sequence encoding an oil-body-targeting-protein operatively linked in reading frame to;

(b) a second nucleic acid sequence encoding a first recombinant polypeptide;

linked in reading frame to;

(c) a third nucleic acid sequence encoding a second recombinant polypeptide, wherein said first and second recombinant polypeptide are capable of forming a multimeric-protein-complex, and

wherein said first recombinant polypeptide is a thioredoxin and said second recombinant polypeptide is a thioredoxin-reductase.

5. A recombinant multimeric-fusion-protein comprising (i) an oil-body-targeting-protein, or fragment thereof, (ii) a first recombinant polypeptide and a (iii) second recombinant polypeptide, wherein said first and second recombinant polypeptides are capable of forming a multimeric-protein-complex, and

wherein said first recombinant polypeptide is a thioredoxin and said second recombinant polypeptide is a thioredoxin-reductase.

6. Isolated oil bodies comprising a multimeric-protein-complex comprising (i) an oil-body-targeting-protein and (ii) a first recombinant polypeptide, said oil bodies further comprising a second recombinant polypeptide, wherein said first and second recombinant polypeptide are capable of forming a multimeric-protein-complex, and

wherein said first recombinant polypeptide is a thioredoxin and said second recombinant polypeptide is a thioredoxin-reductase.

7. Isolated oil bodies comprising

(a) a first fusion protein comprising a first oil-body-targeting-protein fused to a first recombinant polypeptide;

and

(b) a second fusion protein comprising a second oil-body-targeting-protein fused to a second recombinant polypeptide,

wherein said first and second recombinant polypeptide are capable of forming a multimeric-protein-complex, and

wherein said first recombinant polypeptide is a thioredoxin and said second recombinant polypeptide is a thioredoxin-reductase.

12. A method of reducing allergenicity of a food comprising the steps of:

providing the isolated oil bodies of claim 7;

and

adding the isolated oil bodies to the food, whereby allergenicity of the food is reduced.

8. A cell comprising oil bodies and (i) an oil-body-targeting-protein, (ii) a first recombinant polypeptide and (iii) a second recombinant polypeptide wherein

(1) said first recombinant polypeptide is capable of associating with said oil-body-targeting-protein;

and

(2) said first recombinant polypeptide capable of associating with said second recombinant polypeptide to form a multimeric-protein-complex,

wherein said first recombinant polypeptide is a thioredoxin and said second recombinant polypeptide is a thioredoxin-reductase.

9. A composition comprising isolated oil bodies, thioredoxin and thioredoxin-reductase.

10. A food product, personal care product or pharmaceutical composition comprising the composition of claim 9.

11. The personal care product of claim 10 wherein said personal care product reduces the oxidative stress to the surface area of the human body or is used to lighten the skin.

14. A method for preparing an enzymatically active redox protein associated with oil bodies comprising:

a) producing in a cell a redox fusion polypeptide comprising a first redox protein linked to a second redox protein;

b) associating said redox fusion polypeptide with oil bodies through an oil-body-targeting-protein capable of associating with said redox fusion polypeptide and said oil bodies; and

c) isolating said oil bodies associated with said redox fusion polypeptide,

wherein said first redox protein is a thioredoxin and said second redox protein is a thioredoxin-reductase.

15. A method for preparing a redox protein associated with oil bodies comprising:

a) introducing into a cell a chimeric nucleic acid sequence comprising;

1) a first nucleic acid sequence capable of regulating transcription in said cell operatively linked to;

2) a second nucleic acid sequence encoding a recombinant fusion polypeptide comprising (i) a nucleic acid sequence encoding a sufficient portion of an oil-body-protein to provide targeting of said recombinant fusion polypeptide to an oil body linked to (ii) a nucleic acid sequence encoding a redox fusion polypeptide comprising a first redox protein linked to a second redox protein operatively linked to;

3) a third nucleic acid sequence capable of terminating transcription in said cell;

b) growing said cell under conditions to permit expression of said redox fusion polypeptide in a progeny cell comprising oil bodies; and

c) isolating from said progeny cell said oil bodies comprising said redox fusion polypeptide,

wherein said first redox protein is a thioredoxin and said second redox protein is a thioredoxin-reductase.

16. A chimeric nucleic acid comprising:

1) a first nucleic acid sequence capable of regulating transcription in a host cell operatively linked to;

2) a second nucleic acid sequence encoding a recombinant fusion polypeptide comprising (i) a nucleic acid sequence encoding a sufficient portion of an oil-body-protein to provide targeting of said recombinant fusion polypeptide to an oil body linked to (ii) a nucleic acid sequence encoding a redox fusion polypeptide comprising a first redox protein linked to a second redox protein operatively linked to;

3) a third nucleic acid sequence capable of terminating transcription in said cell,

wherein said first redox protein is a thioredoxin and said second redox protein is a thioredoxin-reductase.

17. A transgenenic plant comprising the chimeric nucleic acid sequence of claim 16.

18. A plant seed comprising the chimeric nucleic acid of claim 16.

19. A safflower seed comprising the chimeric nucleic acid of claim 16.

20. A nucleic acid construct comprising a gene fusion, wherein the gene fusion comprises a first region encoding an oil-body-protein or an active fragment thereof, operably linked to a second region encoding at least one thioredoxin-related protein or an active fragment thereof,

wherein the at least one thioredoxin-related protein is selected from thioredoxin or thioredoxin-reductase.

21. A transgenic plant containing a nucleic acid construct comprising a gene fusion, wherein the gene fusion comprises a region encoding an oil-body-protein or an active fragment thereof, operably linked to a region encoding a first thioredoxin-related protein or an active fragment thereof,

wherein the thioredoxin-related protein is selected from thioredoxin or thioredoxin-reductase.

22. A transgenic plant comprising a nucleic acid construct comprising a seed-specific promoter operably linked to a gene fusion, wherein the gene fusion comprises a region encoding an oil-body-protein or an active fragment thereof, operably linked to a region encoding a first thioredoxin-related protein or an active fragment thereof, wherein a fusion protein comprising activities of oleosin and the thioredoxin-related protein is produced in a seed of the plant,

wherein the thioredoxin-related protein is selected from thioredoxin or thioredoxin-reductase.

23. A method of making a fusion protein comprising a thioredoxin-related activity, the method comprising the steps of:

providing a transgenic plant comprising a nucleic acid construct comprising a seed-specific promoter operably linked to a gene fusion, wherein the gene fusion comprises a region encoding an oil-body-protein or an active fragment thereof, operably linked to a region encoding a first thioredoxin-related protein or an active fragment thereof, the gene fusion encoding a fusion protein comprising a thioredoxin-related activity;

obtaining seeds from the plant; and

recovering the fusion protein by isolating oil bodies from the seeds,

wherein the thioredoxin-related protein is selected from thioredoxin or thioredoxin-reductase.

24. A method of reducing allergenicity of a food comprising the steps of:

providing a preparation comprising oil bodies associated with a fusion protein, the fusion protein comprising an oil-body-protein or an active fragment thereof and a thioredoxin-related protein or an active fragment thereof; and

adding the preparation to the food, whereby allergenicity of the food is reduced due to activity of the thioredoxin-related protein or fragment,

wherein the thioredoxin-related protein is selected from thioredoxin or thioredoxin-reductase.

25. A composition comprising a fusion protein, the fusion protein comprising an oil-body-protein or an active fragment thereof and a thioredoxin-related protein or an active fragment thereof, in a pharmaceutically acceptable carrier,

wherein the thioredoxin-related protein is selected from thioredoxin or thioredoxin-reductase.

26. A cosmetic formulation comprising oil bodies associated with a fusion protein, the fusion protein comprising an oil-body-protein or an active fragment thereof and a thioredoxin-related protein or an active fragment thereof, in an acceptable carrier,

wherein the thioredoxin-related protein is selected from thioredoxin or thioredoxin-reductase.

27. A method of treating or protecting a target against oxidative stress, comprising the steps of:

providing a preparation comprising a fusion protein, the fusion protein comprising an oil-body-protein or an active fragment thereof and a thioredoxin-related protein or an active fragment thereof; and

contacting the preparation with a target, wherein the target is susceptible to oxidative stress, thereby treating or protecting against the stress,

wherein the thioredoxin-related protein is selected from thioredoxin or thioredoxin-reductase.

28. A nucleic acid construct comprising a gene fusion, wherein the gene fusion comprises a first region encoding an oil-body-protein or an active fragment thereof, operably linked to a second region encoding at least one recombinant polypeptide and an oil-body-surface-avoiding linker in frame between the first and second region polypeptides.