MODIFIED VIRD2 PROTEIN AND ITS USE IN IMPROVED GENE TRANSFER
First Claim
1. A method of Agrobacterium-mediated gene transformation of a host cell, said Agrobacterium having a VirD2 protein wherein said Agrobacterium has a modified VirD2 protein which is resistant to cleavage by a caspase.
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Abstract
There is provided a method for Agrobacterium-mediated gene transformation of a host cell. The method uses an Agrobacterium which expresses a VirD2 protein resistant to cleavage by a caspase. The VirD2 protein is modified by replacement of an aspartic acid residue with an alternative amino acid at one or more of the caspase cleavage sites within the protein amino acid sequence. This method improves the low efficiency of Agrobacterium-mediated gene transfer in certain plant cells and in animal cells caused by caspase cleavage of the VirD2 protein. The modified VirD2 protein, a gene for its expression and a method of producing a protein of interest by Agrobacterium-mediated transformation of a host cell are also described.
21 Citations
22 Claims
- 1. A method of Agrobacterium-mediated gene transformation of a host cell, said Agrobacterium having a VirD2 protein wherein said Agrobacterium has a modified VirD2 protein which is resistant to cleavage by a caspase.
- 11. An Agrobacterium vector which expresses a VirD2 protein which is resistant to cleavage by caspases.
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13. (canceled)
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14. (canceled)
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15. A VirD2 protein modified by replacement of the D residue of a cleavage site PVTD (SEQ ID NO:
- 3), VNLD (SEQ ID NO;
4), ASLD SEQ ID NO;
5) or DEVD (SEQ ID NO;
6) with an alternative amino acid. - View Dependent Claims (16, 17, 18, 19, 21)
- 3), VNLD (SEQ ID NO;
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20. (canceled)
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22. A method of producing a protein of interest, said method comprising:
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providing an Agrobacterium vector encoding a VirD2 protein modified to have at least one aspartic acid (D) residue replaced by an alternative amino acid so that the modified VirD2 protein is resistant to cleavage by a caspase, and further comprising a gene encoding a protein of interest within the T-DNA thereof; infecting a host cell with said Agrobacterium under conditions suitable to allow stable integration of the gene encoding the protein of interest into the genome of said host cell; and cultivating said host cell or progeny thereof under conditions suitable to allow expression of said protein of interest.
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Specification