Claims for Patent: 9,371,533

Summary for Patent: 9,371,533

Title:	Protein expression system
Abstract:	Disclosed is a DNA polynucleotide comprising a nucleic acid sequence having promoter activity in a Drosophila S2 cell, where said nucleic acid sequence is selected from (i) a nucleotide sequence of SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO: 5, SEQ ID NO:6, SEQ ID NO:33, SEQ ID NO:36, SEQ ID NO: 37, or SEQ ID NO: 68 with or without flanking restriction site sequences at either terminus; (ii) a functional nucleotide sequence with a sequence identity of at least 80% to any one sequence of (i); (iii) a nucleotide which is a functional fragment of at least 6 contiguous nucleotides of any one sequence of (i) or (ii); (iv) a functional nucleotide sequence with a sequence identity of at least 80% to said functional fragment of (iii); (v) a first chimeric nucleotide sequence comprising two or more sequences of any one sequence of (i), (ii), (iii) and (iv), and (vi) a second chimeric nucleotide sequence, comprising at least 6 nucleotides and including consecutive nucleotide stretches from at least 2 nucleotide sequences of (iii) and/or (iv), where each of said consecutive stretches alone does not have promoter activity in a Drosophila S2 cell. Also disclosed are vectors and cells comprising the polynucleotide and a method for producing a polypeptide of interest by use of the polynucleotide.
Inventor(s):	Dyring; Charlotte (Rungsted Kyst, DK), De Jongh; Willem Adriaan (Valby, DK), Rasmussen; Peter Birk (Holte, DK), Lykkegaard; Helene (Hillerod, DK)
Assignee:	Expres2ion Biotechnologies ApS (Horsholm, DK)
Application Number:	12/997,072
Patent Claims:	1. An isolated DNA polynucleotide suitable for heterologous expression of a polypeptide of interest in an insect cell, said DNA polynucleotide comprising a promoter DNA polynucleotide comprising a chimeric sequence comprising residues 7-586 of SEQ ID NO: 68, SEQ ID NO: 68, or the nucleotide sequence SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 33, wherein in each of SEQ ID NOs: 3-6 and 33, having SEQ ID NO: 36 found therein, has SEQ ID NO: 36 substituted by SEQ ID NO: 37. 2. The isolated DNA polynucleotide according to claim 1, wherein said promoter DNA polynucleotide exhibits an increased protein expression level as compared to the protein expression level of any one of the promoter DNA polynucleotides having the sequence of SEQ ID NO:1 or SEQ ID NO:4. 3. The isolated DNA polynucleotide according to claim 2, wherein the increase in protein expression level is from about 50 percent to about 300 percent relative to the protein expression level of any one of the promoter DNA polynucleotides having the sequence of SEQ ID NO:1 or SEQ ID NO:4. 4. The isolated DNA polynucleotide according to claim 2, wherein the increase in protein expression level is 2 fold to 10 fold relative to the protein expression level of any one of the promoter DNA polynucleotides having the sequence of SEQ ID NO:1 or SEQ ID NO:4, or to the pOPIE2 promoter. 5. The isolated DNA polynucleotide according to claim 1, which further comprises a selection marker. 6. The isolated DNA polynucleotide according to claim 5, which further comprises a bacterial promoter. 7. The isolated DNA polynucleotide according to claim 5, which further comprises a second promoter DNA polynucleotide suitable to drive the expression of the selection marker in an insect cell. 8. The isolated DNA polynucleotide according to claim 5, wherein the selection marker is selected from the group consisting of a Zeocin selection marker, a Neomycin selection marker, a Hygromycin selection marker, a Puromycin selection marker, and a Blasticidin selection marker. 9. The isolated DNA polynucleotide according to claim 6, wherein the bacterial promoter is a pKANR bacterial promoter. 10. The isolated DNA polynucleotide according to claim 7, wherein said second promoter is selected from the group consisting of a nucleotide sequence of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:33, SEQ ID NO:36, SEQ ID NO:37, or a nucleotide sequence comprising residues 7-586 of SEQ ID NO: 68. 11. The isolated DNA polynucleotide according to claim 1, which further comprises at least one ubiquitous chromatin opening element upstream and/or downstream relative to a multiple cloning site. 12. The isolated DNA polynucleotide according to claim 1, which further comprises at least one Transcriptional insulator element. 13. The isolated DNA polynucleotide according to claim 12, wherein the transcriptional insulator element is a Gypsy (gsu(Hw)) insulator sequence. 14. The isolated DNA polynucleotide according to claim 1, which further comprises a dihydrofolate reductase (dhfr) coding sequence suitable for selection in insect cells. 15. The isolated DNA polynucleotide according to claim 1, which further comprises at least one polyadenylation signal sequence. 16. The isolated DNA polynucleotide according to claim 15, wherein the polyadenylation sequence is selected from a SV40 polyA signal, an OPIE2 polyA signal, and a combined SV40 polyA signal and OPIE2 polyA signal. 17. The isolated DNA polynucleotide according to claim 1, which further comprises an E. coli origin of replication. 18. The isolated DNA polynucleotide according to claim 1, which further comprises at least one protein export signal polynucleotide sequence. 19. The isolated DNA polynucleotide according to claim 18, wherein the protein export sequence is selected from BIP and CPY. 20. The isolated DNA polynucleotide according to claim 1, which is essentially free of viral DNA. 21. The isolated DNA polynucleotide according to claim 1, which further comprises at least one HIS-tag sequence. 22. The isolated DNA polynucleotide according to claim 1, which further comprises a multiple cloning site downstream of said promoter DNA polynucleotide for insertion of the gene encoding a polypeptide of interest into said isolated DNA polynucleotide. 23. The isolated DNA polynucleotide according to claim 22, comprising the intron shown in SEQ ID NO: 60 including or excluding the flanking restriction sites. 24. The isolated DNA polynucleotide according to claim 1, which further comprises at least one 72 bp element from SV40. 25. The isolated DNA polynucleotide according to claim 1, which further comprises at least one amplification control element. 26. The isolated DNA polynucleotide according to claim 1, which further comprises at least one Ori-beta element. 27. The isolated DNA polynucleotide according to claim 1, which further comprises at least one matrix attachment region (MAR) element. 28. The isolated DNA polynucleotide according to claim 1, which further comprises at least one PRE element from Hepatitis B virus. 29. The isolated DNA polynucleotide according to claim 28, comprising the a PRE element from Hepatitis B virus according to SEQ ID NO:40. 30. The isolated DNA polynucleotide according to claim 28, comprising nucleotides 10 to 574 of SEQ ID NO:40. 31. The isolated DNA polynucleotide according to claim 1, wherein the promoter DNA polynucleotide sequence controls the expression of a gene or other DNA sequence to which it is linked. 32. The isolated DNA polynucleotide according to claim 1, which further comprises at least one intron downstream of the promoter DNA polynucleotide sequence and upstream of a multiple cloning site. 33. The isolated DNA polynucleotide according to claim 1, which is adapted for ligation independent cloning (LIC). 34. The isolated DNA polynucleotide according to claim 1, which further comprises at least one polynucleotide sequence encoding a polypeptide heterologous to said promoter DNA polynucleotide sequence. 35. An isolated cell comprising the isolated DNA polynucleotide according to claim 1. 36. The isolated cell according to claim 35, which is an insect cell. 37. The isolated cell according to claim 35, which is stably transfected with said isolated DNA polynucleotide. 38. The isolated cell according to claim 37, which is a Drosophila melanogaster cell. 39. A method for the production of a polypeptide of interest encoded by a polynucleotide the method comprising the steps of (a) obtaining a polynucleotide sequence encoding the polypeptide of interest; (b) inserting said polynucleotide sequence encoding the polypeptide of interest into the isolated DNA polynucleotide according to claim 1; (c) transforming a host cell with the polynucleotide obtained under step (b); (d) allowing for the expression of said polynucleotide obtained under step (b) to produce the polypeptide; and (e) obtaining the polypeptide there from.

Details for Patent 9,371,533

Applicant	Tradename	Biologic Ingredient	Dosage Form	BLA	Approval Date	Patent No.	Expiredate
Merck Sharp & Dohme Corp.	INTRON A	interferon alfa-2b	For Injection	103132	06/04/1986		2028-06-12
Merck Sharp & Dohme Corp.	INTRON A	interferon alfa-2b	For Injection	103132			2028-06-12
Merck Sharp & Dohme Corp.	INTRON A	interferon alfa-2b	Injection	103132			2028-06-12