Method for agrobacterium mediated transformation of cambial meristematic plant cells to produce recombinant protein
Plant cells express a target protein, a production method thereof, and a method of producing a target protein using the same are disclosed. The plant cells have introduced therein a recombinant vector containing gene(s) encoding a target protein, and the plant cells include plant cambial meristematic cells (CMC) or callus. The cambial meristematic cells (CMCs) are a cell line containing innately undifferentiated cells isolated from a plant, wherein the cell line is a cell line isolated from the cambial tissue of the plant and has meristematic continuity without going through dedifferentiation into callus. A system of expressing a target protein using the recombinant plant cells according to the present invention can overcome the problems of conventional plant cell culture. In addition, it shows significantly high transformation efficiency, and thus can produce large amounts of target proteins, including biopharmaceutical proteins.
1. A method of producing a protein, comprising:
mixing, in a culture, tomato cambial meristematic cells (CMCs) and Agrobacteria that contain a vector comprising a gene encoding the protein, wherein the CMCs are a cell line containing innately undifferentiated cells and having meristematic continuity without going through dedifferentiation into callus;
co-culturing the CMCs along with Agrobacteria containing the vector in the culture while shaking the culture for 1 minute to 48 hrs;
subsequently co-culturing the CMCs and Agrobacteria containing the vector in the culture without shaking the culture, for sufficient time to sediment the CMCs in the culture to have a higher ratio of transfection of the gene encoding the protein than CMCs which are not sedimented;
subsequently continuing to co-culture the CMCs and Agrobacteria containing the vector in the culture while shaking the culture;
wherein said co-culturing of the CMCs and Agrobacteria containing the vector is carried out for a period of 1 to 9 days, and wherein during co-culturing of the CMCs and Agrobacteria containing the vector, the vector is introduced in the CMCs; and
isolating the protein expressed in the culture.
2. The method of claim 1 , wherein during co-culturing of the CMCs and Agrobacteria containing the vector, the gene encoding the protein is transfected in at least part of the CMCs so that the protein is transiently expressed in at least part of the CMCs.
3. The method of claim 1 , wherein during co-culturing of the CMCs and Agrobacteria containing the vector, the gene encoding the protein is stably transformed into at least part of the CMCs.
4. The method of claim 1 , further comprising co-culturing the CMCs and Agrobacteria containing the vector in the culture without shaking of the culture and with shaking of the culture such that co-culturing without shaking is intermittently performed between co-culturing with shaking.
5. The method of claim 1 , wherein the culture containing Agrobacteria has an OD 600 not exceeding 2.0.