Protein expression in plants

Patent metadata

Field	Value
Publication number	US-10472644-B2
Application number	US-201213980099-A
Country	US
Kind code	B2
Filing date	Jan 17, 2012
Priority date	Jan 17, 2011
Publication date	Nov 12, 2019
Grant date	Nov 12, 2019

Abstract

The present invention relates to protein expression in plants, particularly the large-scale production of recombinant polypeptides in whole Nicotiana tabacum plants. The use of preselected combination of N. tabacum varieties and Agrobacterium strains, optionally including one or more improvements to the transient expression-based methods of the invention, enables the production of large quantities of a heterologous polypeptides economically and in a short period of time.

First claim

The invention claimed is:

1. A transient expression based method for producing large quantities of a heterologous polypeptide in Nicotiana tabacum comprising the steps of:
(i) infiltrating a whole plant of Nicotiana tabacum line PM132, seeds of which were deposited under accession number NCIMB 41802 strain with a suspension of the strain of Agrobacterium tumefaciens strain AGL1 or of Agrobacterium tumefaciens strain EHA105 at an OD 600 of between 0.1 and 4.0, the strain comprising an expressible nucleotide sequence encoding the heterologous polypeptide; and
(ii) incubating the infiltrated plant for a period of between 5 days and 10 days under conditions that allow expression of the expressible nucleotide sequence in the infiltrated plant and accumulation of the heterologous polypeptide;
such that approximately 700 mg TurboGFP/kg (tGFP/kg) frozen leaf weight can be obtained in Nicotiana tabacum plants of line PM 132 when using an assay system comprising Agrobacterium strains AGL1 and EHA105, respectively, harboring a gene construct comprising the tGFP gene cloned under the control of the cauliflower mosaic virus 35S promoter and HT-CPMV sequence and the NOS terminator sequence, under the proviso that Agrobacterium strain AGL1 and EHA105, respectively, does not contain a virus expression vector.

2. The method according to claim 1 , wherein the expressible nucleotide sequence encoding the polypeptide is cloned in a minimally-sized binary vector which has a polynucleotide sequence of SEQ ID NO: 1.

3. The method of claim 1 , wherein the suspension of Agrobacterium cells used in step (i) for infiltrating Nicotiana tabacum line PM132, seeds of which were deposited under accession number NCIMB 41802, has a cell density (OD 600 ) in the range of 0.6 to 0.9.

4. The method of claim 1 , wherein a suppressor of gene silencing is transiently expressed in the N. tabacum plant when the nucleotide sequence encoding the heterologous polypeptide is expressed.

5. The method of claim 4 , wherein the nucleotide sequence encoding a suppressor of gene silencing is located on a first binary vector and the nucleotide sequence encoding the heterologous polypeptide is located on a second binary vector.

6. The method of claim 1 , wherein a helper-component proteinase (HcPro) of a potyvirus is transiently expressed in the N. tabacum plant when the nucleotide sequence encoding the heterologous polypeptide is expressed.

7. The method of claim 6 , wherein a helper-component proteinase (HcPro) of a potyvirus is transiently expressed in the N. tabacum plant using a second binary vector that is separate from the first binary vector comprising the nucleotide sequence encoding the heterologous polypeptide.

8. The method of claim 6 , wherein the helper-component proteinase (HcPro) of a potyvirus is encoded by a nucleic acid molecule comprising the nucleotide sequence set forth in SEQ ID NO: 5.

9. The method claim 4 , wherein the first binary vector is provided in a first Agrobacterium strain and the second vector is provided in a second Agrobacterium strain and wherein in step (i), the ratio of cells of the first Agrobacterium strain comprising the first binary vector comprising the nucleotide sequence encoding a heterologous protein, to cells of the second Agrobacterium strain comprising the second binary vector, is in a range from 3:1 to 1.6:1.

10. The method of claim 1 , wherein seeds of Nicotiana tabacum line PM132 were deposited under accession number NCIMB 41802 and wherein the seed depositary is NCIMB in Aberdeen, Scotland.

11. The method of claim 1 , wherein the heterologous polypeptide is an influenza haemagglutinin or an immunogenic fragment thereof.

12. The method of claim 1 , wherein the plants are exposed to light prior to infiltration, such that the stomatal conductance is in a range of between 70 μmol m −2 s −1 and 600 μmol m −2 s −1 .

13. The method of claim 1 , wherein step (ii) comprises incubating the plant under daylight conditions for seven to nine hours per day.

14. The method of claim 1 , wherein step (ii) comprises incubating the infiltrated plant in an inverted position.

15. The method of claim 1 , further comprising (a) prior to infiltration, growing whole tobacco plant of N. tabacum line PM132, seeds of which were deposited under accession number NCIMB 41802, at a density of at least 100 plants per square meter, or (b) after infiltration, incubating the infiltrated whole plants at a density of at least 100 plants per square meter, or (c) prior to infiltration, growing whole tobacco plant of N. tabacum line PM132, seeds of which were deposited under accession number NCIMB 41802, at a density of at least 100 plants per square meter, and after infiltration, incubating the infiltrated whole plants at a density of at least 100 plants per square meter.

16. The method of claim 15 , wherein the plants are grown in a density of between 200 and 600 plants per square meter.

17. The method of claim 1 , further comprising after step (ii), a step (iii) of infiltrating the whole Agrobacterium -infiltrated plant with one or more enzymes that degrade plant cell wall.

18. The method of claim 1 , wherein step (i) comprises infiltrating the whole plant by one or more pressure cycle(s) wherein at least one of the pressure cycle(s) comprises an increase in pressure relative to atmospheric pressure.

19. The method of claim 1 , wherein the expressible nucleotide sequence encoding the polypeptide is cloned in a binary vector comprising a T-DNA region that comprises one or two or more copies of a FLt promoter or a functional fragment thereof, wherein the FLt promoter is that of MMV, FMV or PCISV.

20. The transient expression-based method of claim 1 , wherein the gene construct comprises a TurboGFP gene cloned under control of a cauliflower mosaic virus 35S promoter and HT-CPMV sequence and a NOS terminator sequence.

Assignees

• Beuchat Julien
• Campanoni Prisca
• Dai Shunhong
• Facchinetti Claudio
• Lugon Moulin Nicolas
• Oishi Karen
• Ramirez Gustavo
• Roesti Sandrine
• Laparra Helene
• Cabrera Rosa
• Philip Morris Products Sa

Inventors

• Beuchat Julien
• Campanoni Prisca
• Dai Shunhong
• Facchinetti Claudio
• Lugon-Moulin Nicolas
• Oishi Karen
• Ramirez Gustavo
• Roesti Sandrine
• Laparra Helene
• Cabrera Rosa

Classifications

• C12N15/8257 Primary

  for the production of primary gene products, e.g. pharmaceutical products, interferon · CPC title

• C12N15/8205

  Agrobacterium mediated transformation · CPC title

• C12N15/82

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

• C07K14/005

  from viruses · CPC title

• A01H5/00

  Angiosperms, i.e. flowering plants, characterised by their plant parts; Angiosperms characterised otherwise than by their botanic taxonomy · CPC title