Methods for producing proteins in plants

Patent metadata

Field	Value
Publication number	US-9512439-B2
Application number	US-201113809995-A
Country	US
Kind code	B2
Filing date	Jul 15, 2011
Priority date	Jul 16, 2010
Publication date	Dec 6, 2016
Grant date	Dec 6, 2016

Abstract

The present invention relates to methods for expressing proteins of interest, particularly pharmaceutically valuable proteins, transiently in plants. In particular, the invention provides an improved method for introducing Agrobacterium cells into a whole plant or a plant organ. The methods of the invention provides efficient agroinfiltration of many plants singly or simultaneously resulting in a yield of recombinant proteins that is higher than that obtained by other methods. The methods can be readily scaled and automated to meet changing demands of the recombinant protein.

First claim

The invention claimed is:

1. A system for infiltrating Agrobacterium bacteria into a whole and intact plant or a part of a whole and intact plant and/or for producing a heterologous peptide or protein in a whole and intact plant or a part of a whole and intact plant comprising:
a) a chamber defining an interior and comprising
(i) an inlet in communication with the interior of the chamber;
(ii) an opening lined with an elastic seal;
(iii) an outlet in communication with the interior of the chamber; and

b) a source of compressed air operably coupled to the inlet,
wherein said chamber is configured for receiving through the opening all or part of areal parts of a plant and wherein the elastic seal is configured such that a seal can be formed around the main stem of the plant such that all or part of the aerial parts of a plant is positioned inside the chamber.

2. The system of claim 1 , comprising a compressor and a pressure reducer configured to subject pressure to the whole and intact plant or a part of the whole and intact plant and Agrobacterium cells in suspension during one or more pressure cycle(s).

3. The system of claim 1 , wherein the chamber comprises, in the interior, a plurality of nozzles that are dimensioned for atomizing or aerosolizing a liquid.

4. The system of claim 2 , wherein the chamber is configured such that the entire plant body including its aerial and underground parts is submerged in the infiltration medium containing the Argobacterium cells and subjected to the pressure applied during the one or more pressure cycle(s).

5. The system of claim 2 , wherein the chamber is configured such that only all or part of the aerial parts of the plant are submerged in the infiltration medium containing the Argobacterium cells, but the entire plant including the aerial parts as well as the underground parts of the plant is subjected to the pressure applied during the one or more pressure cycle(s).

6. The system of claim 2 , wherein the chamber is configured such that all or part of the aerial parts of the plant are submerged in the infiltration medium containing the Argobacterium cells and exposed to pressure in the one or more pressure cycles, whereas the underground parts of the plant, particularly the plant root, are positioned outside of the chamber such that they are not submerged in the infiltration medium containing the Agrobacterium cells and not subjected to the pressure applied during the one or more pressure cycle(s).

7. The system of claim 1 , wherein said elastic seal is an elastic pneumatic or hydraulic seal.

8. Use of a system according to claim 1 for infiltrating Agrobacterium bacteria into a whole plant or a plant part and/or for producing a heterologous peptide or protein in a whole plant or a plant part comprising:
a) contacting within the chamber the aerial parts of a whole and intact plant, or a part of the aerial parts of a whole and intact plant, with Agrobacterium cells suspended in a fluid, wherein the Agrobacterium cells comprise an expressible construct encoding the heterologous peptide or protein of interest;
b) treating the aerial parts of the whole and intact plant or a part of the aerial parts of the whole and intact plant and the Agrobacterium cells with one or more pressure cycle(s).

9. The use according to claim 8 , wherein the expressible construct is a plant expressible construct and is selected to provide transient expression of the heterologous peptide or protein of interest.

10. The use according to claim 8 further comprising applying pressure to the whole and intact plant or a part of the whole and intact plant, while said plant or plant part is in contact with the Agrobacterium cells in the bacterial cell suspension.

11. The use according to claim 8 , wherein the suspension of Agrobacterium cells comprises an OD600 of at least 1.0.

12. The use according to claim 8 , wherein the chamber is configured such that the entire plant body including its aerial and underground parts is submerged in the infiltration medium containing the Argobacterium cells and subjected to the pressure applied during the one or more pressure cycle(s).

13. The use according to claim 8 , wherein the chamber is configured such that only all or part of the aerial parts of the plant is submerged in the infiltration medium containing the Argobacterium cells, but the entire plant including the aerial parts as well as the underground parts of the plant is subjected to the pressure applied during the one or more pressure cycle(s).

14. The use according to claim 8 , wherein the chamber is configured such that all or part of the aerial parts of the plant are submerged in the infiltration medium containing the Argobacterium cells and exposed to pressure in the one or more pressure cycles, whereas the underground parts of the plant, particularly the plant root, are positioned outside of the chamber such that they are not submerged in the infiltration medium containing the Agrobacterium cells and not subjected to the pressure applied during the one or more pressure cycle(s).

15. The use according to claim 8 , wherein the step of treating the whole plant or the plant part comprises
a) at least 2 cycles; or
b) at least 3 cycles; or
c) at least 4 cycles; or
d) at least 8 cycles.

16. The use according to claim 8 , wherein at least one of the pressure cycles comprises treating the whole plant or the plant part with a pressure of
a) at least 3.5 bar; or
b) at least 4.5 bar; or
c) at least 6.5 bar; or
d) at least 8 bar; or
e) at least 12 bar.

17. The use according to claim 8 , wherein the pressure is applied for between
a) 0.5 seconds/cycle and 10 seconds/cycle; or
b) 1 second/cycle and 5 seconds/cycle; or
c) 0.5 seconds/cycle and 1 second/cycle.

18. The use according to claim 8 , wherein the step of treating the whole plant or the plant part with a pressure that is increased relative to atmospheric pressure comprises
a) at least 1 cycle, at a pressure of at least 8 bar, for at least 0.5 seconds/cycle; or
b) at least 2 cycles, at a pressure of at least 6 bar, for at least 0.5 seconds/cycle; or
c) at least 8 cycles, at a pressure of at least 4.5 bar, for at least 1 second/cycle.

19. The use according to claim 8 , wherein the step of treating the whole plant or the plant part with a pressure that is increased relative to atmospheric pressure comprises at least 8 cycles, at a pressure of at least 4.5 bar, for at least 1 second/cycle.

20. The use according to claim 8 , wherein the step of contacting the whole plant, or the plant part with Agrobacterium cells comprises
a) dipping the plant or a part thereof in a suspension of Agrobacterium cells, wherein the suspension comprises an OD600 of at least 2.5,
b) exposing the plant or a part thereof to an aerosol generated by using a suspension of Agrobacterium cells comprising an OD600 of at least 2.5.

21. The use according to claim 8 , wherein the step of contacting the whole plant, or the plant part with Agrobacterium cells comprises
a) submerging the plant or a part thereof in a suspension of Agrobacterium cells in an enclosed chamber, and
b) applying liquid fluid pressure to the suspension of Agrobacterium cells comprising the submerged plant or part thereof.

22. The use according to claim 8 , wherein the plant is a Nicotiana species, particularly a Nicotiana tabacum species, at a development stage of 8, 9, or 10, wherein the plants have an av

Assignees

• Carraro Andrea
• Faulkner John
• Klipfel Yorick
• Mironov Oleg
• Oishi Karen
• Roesti Sandrine
• Philip Morris Products Sa

Inventors

• Carraro Andrea
• Faulkner John
• Klipfel Yorick
• Mironov Oleg
• Oishi Karen
• Roesti Sandrine

Classifications

• C12N15/82

  for plant cells {, e.g. plant artificial chromosomes (PACs)} · CPC title

• A01G7/00 Primary

  Botany in general · CPC title

• C12N15/8205 Primary

  Agrobacterium mediated transformation · CPC title

• C12N15/8251 Primary

  Amino acid content, e.g. synthetic storage proteins, altering amino acid biosynthesis · CPC title

• C12N15/00

  Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor (mutants or genetically engineered microorganisms, per se C12N1/00, C12N5/00, C12N7/00; new plants per se A01H; plant reproduction by tissue culture techniques A01H4/00; new animals per se A01K67/00; use of medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases, gene therapy A61K48/00) · CPC title