Interferon-alpha polypeptides and conjugates
View Patent ↗The present invention provides interferon-alpha polypeptides and conjugates, and nucleic acids encoding the polypeptides. The invention also includes compositions comprising these polypeptides, conjugates, and nucleic acids; cells containing or expressing the polypeptides, conjugates, and nucleic acids; methods of making the polypeptides, conjugates, and nucleic acids; and methods of using the polypeptides, conjugates, and nucleic acids.
1. An isolated or recombinant polynucleotide comprising a nucleic acid sequence which encodes a polypeptide having the sequence SEQ ID NO: 38 or a sequence which differs from SEQ ID NO: 38 in 1-8 amino acid positions, which
comprises Ala or Leu at position 48 and optionally further comprises a methionine at the N-terminus, which polypeptide exhibits antiviral activity.
2. The polynucleotide of claim 1 , comprising a nucleic acid sequence which encodes a polypeptide comprising the sequence SEQ ID NO:38.
3. The polynucleotide of claim 2 , wherein the nucleic acid sequence is selected from SEQ ID NO:80, SEQ ID NO:81, SEQ ID NO:82, and SEQ ID NO:90.
4. An isolated host cell comprising the nucleic acid of claim 1 .
5. A vector comprising the polynucleotide of claim 1 .
6. The vector of claim 5 , which is an expression vector comprising the polynucleotide operably linked to a promoter.
7. An isolated host cell comprising the vector of claim 6 .
8. A composition comprising the polynucleotide of claim 1 and an excipient.
9. A method for preparing a polypeptide, the method comprising:
providing a culture comprising a host cell, the host cell comprising an expression vector comprising a promoter operably linked to a polynucleotide, the polynucleotide comprising a nucleic acid sequence which encodes a polypeptide having the sequence SEQ ID NO: 38 or a sequence which differs from SEQ ID NO: 38 in 1-8 amino acid positions, which
comprises Ala or Leu at position 48 and optionally further comprises a methionine at the N-terminus; which polvpeptide exhibits antiviral activity, culturing the culture under conditions which permit expression of the polypeptide, and recovering the polypeptide.
10. The method of claim 9 , wherein the host cell is a glycosylating host cell.
11. The method of claim 9 , wherein the host cell is a bacterial host cell.
12. The method of claim 9 , wherein the bacterial host cell is E. coli.
13. The method of claim 12 , wherein the step of recovering the polypeptide comprises:
(a) isolating inclusion bodies (IB) comprising the polypeptide;
(b) solubilizing the IB, thereby obtaining unfolded polypeptide;
(c) refolding the unfolded polypeptide, thereby obtaining refolded polypeptide; and
(d) purifying the refolded polypeptide.
14. The method of claim 13 , wherein the step of solubilizing the IB comprises:
(i) resuspending the IB in 8 M guanidinium chloride, 100 mM dithiothreitol (DTT), and 50 mM Tris pH 8.3;
(ii) mixing the resuspended IB for 30 minutes at room temperature;
(iii) adjusting the pH to 4.5 with HCl; and
(iv) further concentrating the resulting solution by diafiltration using a 5 K membrane against a 10 fold volume of 7.2 M Guanidinium chloride adjusted to pH 4.5 with HCl,
thereby providing a solution of solubilized unfolded polypeptide.
15. The method of claim 14 , wherein the step of refolding the unfolded polypeptide comprises: diluting the solution of solubilized unfolded polypeptide into 0.8 M arginine, 50 mM potassium phosphate, 2 mM reduced glutathione (GSH), 1 mM oxidized glutathione (GSSG) pH 7.8 to a final concentration of about 1 mg/ml polypeptide, and slowly mixing for 2-8° C. overnight, thereby providing a solution of refolded polypeptide.
16. The method of claim 9 , wherein the polypeptide comprises the sequence SEQ ID NO:38.
17. The method of claim 16 , wherein the polynucleotide comprises a nucleic acid sequence selected from SEQ ID NO:80, SEQ ID NO:81, SEQ ID NO:82, and SEQ ID NO:90.
18. An isolated or recombinant polynucleotide comprising a nucleic acid sequence which encodes a polypeptide, said polypeptide comprising the sequence SEQ ID NO:38 or the sequence of a variant thereof wherein the variant differs in 1 to 2 amino acid positions from SEQ ID NO:38, optionally further comprising a methionine at the N-terminus, which polypeptide exhibits antiviral activity.
19. A method for preparing a polypeptide, the method comprising:
providing a culture comprising a host cell, the host cell comprising an expression vector comprising a promoter operably linked to a polynucleotide, the polynucleotide comprising a nucleic acid sequence which encodes a polypeptide comprising the sequence SEQ ID NO:38 or the sequence of a variant thereof wherein the variant differs in 1 to 2 amino acid positions from SEQ ID NO:38, optionally further comprising a methionine at the N-terminus, which polypeptide exhibits antiviral activity,
culturing the culture under conditions which permit expression of the polypeptide, and recovering the polypeptide.
20. The method of claim 19 , wherein the host cell is a glycosylating host cell.
21. The method of claim 19 , wherein the host cell is a bacterial host cell.
22. The method of claim 21 , wherein the bacterial host cell is E. coli.
23. The method of claim 22 , wherein the step of recovering the polypeptide comprises:
(a) isolating inclusion bodies (IB) comprising the polypeptide;
(b) solubilizing the IB, thereby obtaining unfolded polypeptide;
(c) refolding the unfolded polypeptide, thereby obtaining refolded polypeptide; and
(d) purifying the refolded polypeptide.
24. The method of claim 23 , wherein the step of solubilizing the IB comprises:
(i) resuspending the IB in 8 M guanidinium chloride, 100 mM dithiothreitol (DTT), and 50 mM Tris pH 8.3;
(ii) mixing the resuspended IB for 30 minutes at room temperature;
(iii) adjusting the pH to 4.5 with HCI; and
(iv) further concentrating the resulting solution by diafiltration using a 5 K membrane against a 10 fold volume of 7.2 M Guanidinium chloride adjusted to pH 4.5 with HCI, thereby providing a solution of solubilized unfolded polypeptide.
25. The method of claim 24 , wherein the step of refolding the unfolded polypeptide comprises: diluting the solution of solubilized unfolded polypeptide into 0.8 M arginine, 50 mM potassium phosphate, 2 mM reduced glutathione (GSH), 1 mM oxidized glutathione (GSSG) pH 7.8 to a final concentration of about 1 mg/ml polypeptide, and slowly mixing for 2-8° C. overnight, thereby providing a solution of refolded polypeptide.