Recombinant lactoferrins, methods of production from plants and uses thereof
The invention concerns the use of a recombinant nucleotide sequence containing a cDNA coding for a lactoferrin, in particular human lactoferrin, or the derived proteins, and elements enabling a plant cell to produce lactoferrin or the derived proteins, coded by the cDNA, in particular a transcription promoter and terminator identified by the plant cell transcription machinery, to transform plant cells in order to obtain, from these cells, or plants obtained therefrom, lactoferrin or derived proteins.
1. A pharmaceutical composition comprising mature lactoferrin obtained by a method of producing lactoferrin comprising: transforming plant cells using a vector comprising a nucleotide sequence which comprises: (i) a coding sequence for the mature lactoferrin protein and (ii) elements required by a plant cell to produce the lactoferrin coded by the sequence so as to integrate the recombinant sequence within the genome of the cells; and recovering recombinant mature lactoferrin protein produced in the transformed cells, wherein the plant cells are from a monocotyledonous plant or a dicotyledonous plant.
2. The pharmaceutical composition of claim 1 , wherein recovering recombinant mature lactoferrin comprises extracting the lactoferrin from the cells followed by purifying the lactoferrin.
3. A pharmaceutical composition comprising mature lactoferrin obtained by a method of producing mature lactoferrin comprising: (a) transforming plant cells so as to integrate a recombinant nucleotide sequence within the genome of the plant cells, the recombinant nucleotide sequence comprising: (i) a coding sequence for the mature lactoferrin, (ii) a transcription promoter and terminator which are operatively linked to the coding sequence of (i) and are recognized by transcriptional machinery of plant cells, and (iii) a coding sequence for a signal peptide that directs secretions of recombinant peptides from plant cells (b) expressing lactoferrin in said transformed plant cells, and (c) recovering the lactoferrin expressed in said transformed plant cells, wherein the expressed lactoferrin is mature lactoferrin, wherein the plant cells are from a monocotyledonous plant or a dicotyledonous plant.
4. The pharmaceutical composition of claim 3 , wherein the coding sequence for the lactoferrin is a coding sequence for human lactoferrin.
5. The pharmaceutical composition of claim 3 , wherein the coding sequence for a signal peptide is a sequence selected from the group consisting of a coding sequence for a signal peptide of lactoferrin, a coding sequence for the signal peptide of sweet potato sporamin A and a coding sequence for PRS (pathogenesis related protein).
6. The pharmaceutical composition of claim 3 , wherein the terminator is selected from the group consisting of the terminator polyA 35S of the CaMV (cauliflower mosaic virus), and the terminator polyA NOS of Agrobacterium tumefaciens and is located downstream of the coding sequence for the mature lactoferrin.
7. The pharmaceutical composition of claim 3 , wherein the transcription promoter is selected from the group consisting of the 35S promoter of the CaMV, the double constitutive promoter of the CaMV, the promoter pCRU of the radish cruciferin gene, the promoters pGA1 and pGA6 of Arabidopsis thaliana, the chimeric promoter super-promoter PSP of Agrobacterium tumefaciens, the rice actin promoter followed by the actin intron PAR-IRA promoter, the wheat HMWG promoter, and the maize gamma-zein promoter.
8. The pharmaceutical composition of claim 3 , wherein the recovering of the recombinant lactoferrin is by extraction followed by purification.
9. The pharmaceutical composition of claim 3 , wherein the transforming of plant cells is by means of Agrobacterium tumefaciens transformed by a vector containing the recombinant sequence.
10. The pharmaceutical composition of claim 3 , wherein: the coding sequence for lactoferrin is a coding sequence for human lactoferrin, the coding sequence for a signal peptide is a coding sequence for a signal peptide of sweet potato sporamin A, the terminator is the terminator polyA 35S of the CaMV, and the transcription promoter is the wheat HMWG promoter.
11. A pharmaceutical composition comprising lactoferrin obtained by the method of producing mature lactoferrin comprising: (a) transforming plant cells so as to integrate a recombinant nucleotide sequence within the genome of the plant cells and the recombinant nucleotide sequence comprising: (i) the coding sequence for the mature lactoferrin, (ii) a transcription promoter and terminator which are operably linked to the coding sequence of (i) and are recognized by transcriptional machinery of plant cells, and (iii) a coding sequence for a signal peptide that directs secretion of recombinate peptides from plant cells; (b) producing transformed plants from the transformed plant cells; (c) expressing lactoferrin from said transformed plants, and (d) recovering the lactoferrin expressed from said transformed plants, wherein the expressed lactoferrin is mature lactoferrin, wherein the plant cells are from a monocotyledonous plant or a dicotyledonous plant.
12. The pharmaceutical composition of claim 11 , wherein the coding sequence for the mature lactoferrin is a coding sequence for human lactoferrin.
13. The pharmaceutical composition of claim 11 , wherein the coding sequence for the signal peptide is a sequence selected from the group consisting of a coding sequence for a signal peptide of lactoferrin, a coding sequence for the signal peptide of sweet potato sporamin A and a coding sequence for PRS.
14. The pharmaceutical composition of claim 11 , wherein the terminator is selected from the group consisting of the terminator polyA 35S of the CaMV, and the terminator polyA NOS of Agrobacterium tumefaciens and is located downstream of the coding sequence for the mature lactoferrin.
15. The pharmaceutical composition of claim 11 , wherein the transcription promoter is selected from the group consisting of the 35S promoter of the CaMV, the double constitutive promoter of the CaMV, the promoter pCRU of the radish cruciferin gene, the promoters pGA1 and pGA6 of Arabidopsis thaliana, the chimeric promoter super-promoter PSP of Agrobacterium tumefaciens, the rice actin promoter followed by the actin intron PAR-IRA promoter, the wheat HMWG promoter, and the maize gamma-zein promoter.
16. The pharmaceutical composition of claim 11 , wherein the recovering of the recombinant mature lactoferrin is by extraction followed by purification.
17. The pharmaceutical composition of claim 11 , wherein the transforming of plant cells is by means of Agrobacterium tumefaciens transformed by a vector containing the recombinant sequence.
18. The pharmaceutical composition of claim 11 , wherein: the coding sequence for lactoferrin is a coding sequence for human lactoferrin, the coding sequence for a signal peptide is a coding sequence for a signal peptide of sweet potato sporamin A, the terminator is the terminator polyA 35S of the CaMV, and the transcription promoter is the wheat HMWG promoter.
19. A pharmaceutical composition comprising mature lactoferrin obtained by a method of producing lactoferrin comprising: transforming plant cells using a vector comprising a nucleotide sequence which comprises: (i) a coding sequence for the mature lactoferrin protein and (ii) elements required by a plant cell to produce the lactoferrin coded by the sequence so as to integrate the recombinant sequence within the genome of the cell; producing transformed plants from the transformed cells; and recovering recombinant mature lactoferrin protein produced in the transformed plants, wherein the plant cells are from a monocotyledonous plant or a dicotyledonous plant .
20. The pharmaceutical composition of claim 19 , wherein recovering recombinant mature lactoferrin comprises extracting the lactoferrin from the plants followed by purifying the lactoferrin.