TATk-CDKL5 fusion proteins, compositions, formulations, and use thereof
View Patent ↗Disclosed herein are compositions and formulations containing a TATk-CDKL5 fusion protein. Also disclosed are methods of producing a TATk-CDKL5 fusion protein from vectors containing a TATk-CDKL5 cDNA and methods of transducing cells with the vectors containing a TATk-CDKL5 cDNA and the TATk-CDKL5 fusion protein.
1. A fusion protein comprising:
a CDKL5 polypeptide sequence, wherein the CDKL5 polypeptide sequence comprises SEQ ID NO:2 or SEQ ID NO: 16; and
a TATκ polypeptide sequence, wherein the TATκ polypeptide sequence comprises SEQ ID NO: 4, wherein the TATκ polypeptide is operatively coupled to the CDKL5 polypeptide.
2. The fusion protein of claim 1 , further comprising an Igk-chain leader sequence polypeptide, wherein the Igk-chain leader sequence is operatively coupled to the CDKL5 polypeptide.
3. The fusion protein of claim 1 , further comprising a reporter protein polypeptide, wherein the reporter protein polypeptide is operatively coupled to the CDKL5 polypeptide.
4. The fusion protein of claim 1 , further comprising a protein tag polypeptide, wherein the protein tag polypeptide is operatively coupled to the CDKL5 polypeptide.
5. The fusion protein of claim 1 , wherein the fusion protein has a polypeptide sequence comprising SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, or SEQ ID NO: 14.
6. The fusion protein of claim 1 , wherein the fusion protein increases neurite growth, elongation, branch number, or branch density in the brain of a treated subject as compared to an untreated control subject.
7. The fusion protein of claim 1 , wherein the fusion protein reduces neuron apoptosis in the brain of a treated subject as compared to an untreated control subject.
8. A recombinant nucleic acid comprising:
a CDKL5 nucleic acid sequence, wherein the CDKL5 nucleic acid sequence comprises SEQ ID NO: 1 or SEQ ID NO: 15; and
a TATκ nucleic acid sequence, wherein the TATK nucleic acid sequence about 90% to about 100% sequence identity to SEQ ID NO: 3, wherein the TATK nucleic acid sequence is operatively coupled to the CDKL5 nucleic acid sequence.
9. The recombinant nucleic acid of claim 8 , wherein the nucleic acid encodes a fusion protein that increases neurite growth, elongation, branch number, or branch density in the brain of a treated subject as compared to an untreated control subject.
10. The recombinant nucleic acid of claim 8 , wherein the nucleic acid encodes a fusion protein that reduces neuron apoptosis in the brain of a treated subject as compared to an untreated control subject.
11. The recombinant nucleic acid of claim 8 , wherein the recombinant nucleic acid further comprises a nucleic acid sequence corresponding to a protein tag, wherein the nucleic acid sequence corresponding to a protein tag is operatively coupled to the CDKL5 nucleic acid sequence.
12. The recombinant nucleic acid of claim 11 , wherein the protein tag is selected from the group consisting of: chitin binding protein (CBP), maltose binding protein (MBP), glutathione-S-transferase (GST), poly(His), thioredoxin (TRX), poly(NANP), a FLAG-tag, V5-tag, Myc-tag, HA-tag, S-tag, SBP-Tag, Sftag 1, Softag 3, Tc tag, Xpress tag, Strep-tag, Isopeptag, Spy Tag, Ty tag, Biotin Carboxyl Carrier Protein (BCCP), Nus tag, and combinations thereof.
13. The recombinant nucleic acid of claim 8 , wherein the recombinant nucleic acid further comprises a nucleic acid sequence corresponding to a reporter protein, wherein the nucleic acid sequence corresponding to the reporter protein is operatively coupled to the CDKL5 nucleic acid sequence.
14. The recombinant nucleic acid of claim 13 , wherein the reporter protein is selected from the group consisting of: a fluorescent protein, beta-galactosidase, a luciferase protein, an antibiotic-resistance protein, p-glucuronidase, and alkaline phosphatase.
15. A vector comprising a recombinant nucleic acid of claim 8 .
16. The vector of claim 15 , wherein the nucleic acid encodes a fusion protein that increases neurite growth, elongation, branch number, or branch density in the brain of a treated subject as compared to an untreated control subject.
17. The vector of claim 15 , wherein the nucleic acid encodes a fusion protein that reduces neuron apoptosis in the brain of a treated subject as compared to an untreated control subject.
18. The vector of claim 15 , wherein the recombinant nucleic acid further comprises a nucleic acid sequence corresponding to a protein tag, wherein the nucleic acid sequence corresponding to a protein tag is operatively coupled to the CDKL5 nucleic acid sequence.
19. The vector of claim 18 , wherein the protein tag is selected from the group consisting of: chitin binding protein (CBP), maltose binding protein (MBP), glutathione-S-transferase (GST), poly(His), thioredoxin (TRX), poly(NANP), a FLAG-tag, V5-tag, Myc-tag, HA-tag, S-tag, SBP-Tag, Sftag 1, Softag 3, Tc tag, Xpress tag, Strep-tag, Isopeptag, Spy Tag, Ty tag, Biotin Carboxyl Carrier Protein (BCCP), Nus tag, and combinations thereof.
20. The vector of claim 15 , wherein the recombinant nucleic acid further comprises a nucleic acid sequence corresponding to a reporter protein, wherein the nucleic acid sequence corresponding to the reporter protein is operatively coupled to the CDKL5 nucleic acid sequence.
21. The vector of claim 15 , wherein the reporter protein is selected from the group consisting of: a fluorescent protein, beta-galactosidase, a luciferase protein, an antibiotic-resistance protein, p-glucuronidase, and alkaline phosphatase.
22. The vector of claim 15 , wherein the vector is a bacterial vector or an eukaryotic expression vector.
23. A composition comprising a recombinant nucleic acid of claim 8
and a pharmaceutically acceptable carrier.
24. The composition of claim 23 , wherein the nucleic acid encodes a fusion protein that increases neurite growth, elongation, branch number, or branch density in the brain of a treated subject as compared to an untreated control subject.
25. The composition of claim 23 , wherein the nucleic acid encodes a fusion protein that reduces neuron apoptosis in the brain of a treated subject as compared to an untreated control subject.
26. The composition of claim 23 , wherein the recombinant nucleic acid further comprises a nucleic acid sequence corresponding to a protein tag, wherein the nucleic acid sequence corresponding to a protein tag is operatively coupled to the CDKL5 nucleic acid sequence.
27. The composition of claim 26 , wherein the protein tag is selected from the group consisting of: chitin binding protein (CBP), maltose binding protein (MBP), glutathione-Stransferase (GST), poly(His), thioredoxin (TRX), poly(NANP), a FLAG-tag, V5-tag, Myc-tag, HA-tag, S-tag, SBP-Tag, Sftag 1, Softag 3, Tc tag, Xpress tag, Strep-tag, Isopeptag, Spy Tag, Ty tag, Biotin Carboxyl Carrier Protein (BCCP), Nus tag, and combinations thereof.
28. The composition of claim 23 , wherein the recombinant nucleic acid further comprises a nucleic acid sequence corresponding to a reporter protein, wherein the nucleic acid sequence corresponding to the reporter protein is operatively coupled to the CDKL5 nucleic acid sequence.
29. The composition of claim 28 , wherein the reporter protein is selected from the group consisting of: a fluorescent protein, beta-galactosidase, a luciferase protein, an antibiotic-resistance protein, p-glucuronidase, and alkaline phosphatase.
30. The composition of claim 23 , wherein the CDKL5 nucleic acid sequence and the TATK nucleic acid sequence are contained in a vector.
31. A method comprising:
transfecting cells with a recombinant nucleic acid, where the recombinant nucleic acid comprises:
a CDKL5 nucleic acid sequence, wherein the CDKL5 nucleic acid sequence comprises SEQ ID NO: 1 or SEQ ID NO: 15; and
a TATK nucleic acid sequence, wherein the TATK nucleic acid sequence about 90% to about 100% sequence identity to SEQ ID NO: 3, wherein the TATK nucleic acid sequence is operatively coupled to the CDKL5 nucleic acid sequence.
32. The method of claim 31 , wherein the recombinant nucleic acid is contained in a vector.
33. The method of claim 31 , wherein the cells comprise bacterial cells.
34. The method of claim 31 , wherein the cells comprise eukaryotic cells.
35. The method of claim 31 , further comprising the step of culturing the cells in media for a period of time.
36. The method of claim 35 , further comprising the step of collecting the media after the period of time.
37. The method of claim 36 , further comprising the step of harvesting a recombinant polypeptide from the collected media, where the recombinant polypeptide comprises a CDKL5 polypeptide operatively coupled to a TATκ polypeptide produced from the recombinant nucleic acid.
38. The method of claim 35 , further comprising the step of collecting the cells after the period of time.
39. The method of claim 38 , further comprising the steps of lysing the collected cells to form a cell lysate and harvesting a recombinant polypeptide from the cell lysate, where the recombinant polypeptide comprises a CDKL5 polypeptide operatively coupled to a TATK polypeptide produced from the recombinant nucleic acid.