Claims
- 1. A synthetic nucleotide sequence encoding an erythrocyte-binding protein of a malaria pathogen, wherein codon usage of the synthetic nucleotide sequence is altered compared to a naturally occurring sequence of the erythrocyte-binding protein in order to approximate codon usage of a host of the malaria pathogen.
- 2. The synthetic nucleotide sequence of claim 1, wherein the host is a human, and wherein the erythrocyte-binding protein is a Plasmodium falciparum EBA-75 protein.
- 3. The synthetic nucleotide sequence of claim 1, wherein host is a human, and wherein the erythrocyte-binding protein is a region II Plasmodium falciparum EBA-75 protein.
- 4. The synthetic nucleotide sequence of claim 3, wherein the sequence is SEQ ID NO:1 or SEQ ID NO:3.
- 5. The synthetic nucleotide sequence of claim 3, wherein the sequence encodes an amino acid sequence set forth in SEQ ID NO:2 or SEQ ID NO:4.
- 6. The synthetic nucleotide sequence of claim 3, wherein the synthetic nucleotide sequence is altered to remove one or more N-glycosylation sites.
- 7. A substantially purified recombinant erythrocyte-binding protein of a malaria pathogen encoded by a synthetic nucleotide sequence, wherein codon usage of the synthetic nucleotide sequence is altered compared to a naturally occurring sequence of the erythrocyte-binding protein in order to approximate codon usage of a host of the malaria pathogen.
- 8. The substantially purified protein of claim 7, wherein the host is a human, and wherein the erythrocyte-binding protein is a Plasmodium falciparum EBA-75 protein.
- 9. The substantially purified protein of claim 7, wherein the host is a human, and wherein the erythrocyte-binding protein is a region II Plasmodium falciparum EBA-75 protein.
- 10. The substantially purified protein of claim 9, wherein the synthetic nucleotide sequence is SEQ ID NO:1 or SEQ ID NO:3.
- 11. The substantially purified protein of claim 9 having an amino acid sequence set forth in SEQ ID NO:2 or SEQ ID NO:4.
- 12. The substantially purified protein of claim 9, wherein the synthetic nucleotide sequence is altered to remove one or more N-glycosylation sites.
- 13. A pharmaceutical composition for induction of an anti-malarial immune response, comprising at least one of a substantially purified recombinant erythrocyte-binding protein of a malaria pathogen encoded by a synthetic nucleotide sequence, or the synthetic nucleotide sequence, wherein codon usage of the synthetic nucleotide sequence is altered compared to a naturally occurring sequence of the erythrocyte-binding protein in order to approximate codon usage of a host of the malaria pathogen.
- 14. The pharmaceutical composition of claim 13, wherein the host is a human, and wherein the erythrocyte-binding protein is a Plasmodium falciparum EBA-75 protein.
- 15. The pharmaceutical composition of claim 13, wherein the host is a human, and wherein the erythrocyte-binding protein is a region II Plasmodium falciparum EBA-75 protein.
- 16. The pharmaceutical composition of claim 15, wherein the synthetic nucleotide sequence is SEQ ID NO:1 or a SEQ ID NO:3.
- 17. The pharmaceutical composition of claim 15, wherein the erythrocyte-binding protein amino acid sequence is set forth in SEQ ID NO:2 or SEQ ID NO:4.
- 18. The pharmaceutical composition of claim 15, wherein the synthetic nucleotide sequence is altered to remove one or more N-glycosylation sites.
- 19. A method of inducing an anti-malarial immune response in a host of a malaria pathogen, comprising administering to the host of the malaria pathogen an effective amount of a pharmaceutical composition comprising at least one of a substantially purified recombinant erythrocyte-binding protein of the malaria pathogen encoded by a synthetic nucleotide sequence, or the synthetic nucleotide sequence, wherein codon usage of the synthetic nucleotide sequence is altered compared to a naturally occurring sequence of the erythrocyte-binding protein in order to approximate codon usage of the host of the malaria pathogen, and wherein the effective amount is effective to induce the anti-malarial immune response in the host.
- 20. The method of claim 19, wherein the host is a human, and wherein the erythrocyte-binding protein is a Plasmodium falciparum EBA-75 protein.
- 21. The method of claim 19, wherein the host is a human, and wherein the erythrocyte-binding protein is a region II Plasmodium falciparum EBA-75 protein.
- 22. The method of claim 21, wherein the synthetic nucleotide sequence is SEQ ID NO:1 or SEQ ID NO:3.
- 23. The method of claim 21, wherein the synthetic nucleotide sequence is altered to remove one or more N-glycosylation sites.
- 24. The method of claim 21, wherein the erythrocyte-binding protein amino acid sequence is set forth in SEQ ID NO:2 or SEQ ID NO:4.
- 25. A method of optimizing expression in Pichia pastoris of a recombinant Plasmodium falciparum region II EBA-75 erythrocyte-binding protein encoded by a synthetic nucleotide sequence set forth in SEQ ID NO:1 or SEQ ID NO:3, comprising
a) transforming a strain of Pichia pastoris with an expression vector comprising SEQ ID NO:3 or SEQ ID NO:1, or a fragment thereof, thereby generating a transformed Pichia pastoris strain; b) inoculating the transformed Pichia pastoris strain into a fermentation medium, thereby generating an inoculated fermentation medium; c) subjecting the fermentation medium to a fermentation process at a set of fermentation conditions; d) evaluating levels of expression of the region II EBA-75 erythrocyte-binding protein in the fermentation medium; f) changing the fermentation conditions in step c and repeating steps a through e, wherein the fermentation conditions comprise temperature, pH, or cell mass and are changed in this order, and e) comparing the levels of expression of the region II EBA-75 erythrocyte-binding protein among different fermentation conditions.
- 26. A method of obtaining a substantially pure recombinant Plasmodium falciparum region II EBA-75 erythrocyte-binding protein encoded by a synthetic nucleotide sequence set forth in SEQ ID NO:1 or SEQ ID NO:3, comprising
transforming a strain of Pichia pastoris with an expression vector comprising SEQ ID NO:3 or SEQ ID NO:1; inoculating the transformed Pichia pastoris strain into a fermentation medium; subjecting the fermentation medium to a fermentation process; collecting a supernatant of the fermentation medium; and subjecting the supernatant of the fermentation medium to a protein purification process.
- 27. A method of obtaining a DNA vaccine for inducing an anti-malarial immune response, comprising incorporating a synthetic nucleotide sequence encoding an erythrocyte-binding protein of a malaria pathogen, into a DNA vaccine vector, wherein codon usage of the synthetic nucleotide sequence is altered compared to a naturally occurring sequence of the erythrocyte-binding protein in order to approximate codon usage of a host of the malaria pathogen.
- 28. The method of claim 27, wherein the synthetic nucleotide sequence is set forth in SEQ ID NO:1 or SEQ ID NO:3, or a fragment thereof.
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of U.S. Provisional Patent Application Serial No. 60/345,051, filed Nov. 9, 2001.
Government Interests
[0002] The U.S. Government has rights in this invention arising out of National Institutes of Health (NIAID) Advanced Technology Small Business Innovative Research Grant number AI-46209.
Provisional Applications (1)
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Number |
Date |
Country |
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60345051 |
Nov 2001 |
US |