Production of cellulase enzymes in plant hosts using transient agroinfiltration
Described herein are methods useful for producing proteins, such as enzymes, by agrofiltration. The methods involve producing an Agrobacterium with a Ti plasmid encoding a cellulase, infecting plant cells with the Agrobacterium , allowing expression of the cellulase, and recovering the cellulase from the plant cells. In one embodiment, the protein produced is an endoglucanase.
1. A method of producing a full-length cellulase by transient agroinfiltration, the method comprising:
providing an Agrobacterium , the Agrobacterium comprising a Ti plasmid encoding the cellulase;
combining the Agrobacterium with detached plant tissue using transient agroinfiltration to form a mixture and to allow infection of at least one plant cell of detached plant tissue;
recovering a cellulase-containing fraction from the mixture after a period of time sufficient for the detached plant tissue to transiently express the cellulose; and
recovering a functional full-length cellulase from the cellulase-containing fraction.
2. The method of claim 1 , wherein the period of time is at least 4 days.
3. The method of claim 1 , wherein the cellulase is from a thermophilic organism.
4. The method of claim 1 , wherein the cellulase is an exoglucanase.
5. The method of claim 1 , wherein the cellulase is an endoglucanase.
6. The method of claim 5 , wherein the endoglucanase is β-1,4-endoglucanase E1 from Acidothermus cellulolyticus.
7. The method of claim 1 , wherein the cellulase has an activity of at least 40,000 nmol MU/min/kg fresh plant tissue weight at a pH of 5.5 and a temperature of 65° C.
8. The method of claim 1 , wherein the Agrobacterium is A. tumefaciens.
9. The method of claim 1 , wherein recovering the cellulase-containing fraction comprises rupturing the cells of the plant tissue.
10. The method of claim 1 , wherein expression of the cellulase is under the control of a constitutive promoter.
11. The method of claim 10 , wherein the constitutive promoter is 35S from cauliflower mosaic virus.
12. The method of claim 1 , wherein combining the Agrobacterium with detached plant tissue comprises pressure infiltration.
13. The method of claim 1 , wherein combining the Agrobacterium with detached plant tissue comprises vacuum infiltration.
14. The method of claim 1 , wherein the cellulase is thermostable.
15. The method of claim 1 , wherein the plant tissue is from Nicotiana benthamiana.
16. The method of claim 1 , wherein the cellulase is linked to a signal peptide.
17. The method of claim 1 , wherein recovering a cellulase-containing fraction occurs within nine days of combining the Agrobacterium with the detached plant tissue.
18. The method of claim 6 , wherein at least 1 mg cellulase per kg fresh plant cell weight is expressed within 4 days after infection of the at least one plant cell.
19. The method of claim 6 , wherein at least 2.6 mg cellulase per kg fresh plant cell weight is expressed within 6 days after infection of the at least one plant cell.
20. The method of claim 6 , wherein recovering a cellulase-containing fraction occurs within nine days of combining the Agrobacterium with the detached plant tissue.
21. The method of claim 20 , wherein for each kilogram of the plant tissue combined with the Agrobacterium at least 2.6 mg of cellulase are recovered in the cellulase-containing fraction.