Translation enhancer-element dependent vector systems

• US 20070048776A1
• Filed: 08/23/2006
• Published: 03/01/2007
• Est. Priority Date: 08/24/2005
• Status: Active Grant

• Current Assignee: The Scripps Research Institute
• Original Assignee: The Scripps Research Institute
• Inventors: Zhou, Wei, Edelman, Gerald, Mauro, Vincent
• Granted Patent: US 9,068,197 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12N 15/1051 Gene trapping, e.g. exon-, intron-, IRES-, signal sequence-trap cloning, trap vectors
• CPC Class Codes: C12N 15/1086 Preparation or screening of expression libraries, e.g. reporter assays
• CPC Class Codes: C12N 15/67 General methods for enhancing the expression
• CPC Class Codes: C12N 15/85 for animal cells
• CPC Class Codes: C12N 2800/107 for mammalian
• CPC Class Codes: C12N 2830/002 inducible enhancer/promoter combination, e.g. hypoxia, iron, transcription factor
• CPC Class Codes: C12N 2840/105 enhancing translation
• CPC Class Codes: C12N 2840/20 translation of more than one cistron
• CPC Class Codes: C12N 2840/203 having an IRES

Abstract

A translation enhancer-driven positive feedback vector system is disclosed which is designed to facilitate identification of a Translational Enhancer Element (TEE) and to provide a means for overexpression of gene products. The system exploits both transcriptional and translational approaches to control the expression levels of genes and/or gene products. Methods are also disclosed for screening libraries of random nucleotide sequences to identify translational elements and for overproduction of proteins, which have uses in both research and industrial environments.

Claims

1. A nucleic acid vector comprising a first construct comprising two or more first transcriptional elements, a first cistron encoding a transcription factor, and one or more first translational enhancer elements (TEEs), wherein the transcription factor amplifies the transcription of at least one cistron of the first construct.

2. The vector of claim 1, wherein the TEEs are interposed between a transcription start site within the first transcriptional elements and the first cistron.

3. The vector of claim 1, further comprising at least one cistron on one or more second constructs comprising at least one transcriptional unit.

4. The vector of claim 1, wherein the first construct and the at least one transcriptional unit of one or more second constructs comprise transcriptional elements that are targets for the transcription factor encoded by the first cistron.

5. The vector of claim 1, wherein at least one of the sequences comprising the first transcriptional elements is not the target of transcription factors endogenous to a host cell.

6. The vector of claim 1, wherein the transcription factor is not endogenous to a host cell.

7. The vector of claim 3, wherein at least one cistron of the one or more second constructs encodes at least one gene product.

8. The vector of claim 7, wherein the gene product is a reporter protein, therapeutic protein an enzyme, an antigen, an antibody, or a structural protein.

9. The vector of claim 8, wherein the reporter protein is GFP, luciferase, β

-galactosidase, β

-glucuronidase, alkaline phosphatase, chloramphenicol acetyltransferase, ECFP, EGFP, or EYFP.

10. The vector of claim 7, wherein at least one second construct comprises at least one transcriptional element and one or more TEEs.

11. The vector of claim 10, wherein the at least one second construct comprises one or more TEEs interposed between a transcription start site within the at least one transcriptional element and the cistron encoding the gene product.

12. The vector of claim 7, wherein at least one gene product blocks host protein synthesis.

13. The vector of claim 12, wherein the gene product is NSP3, L-proteinase, or proteinase 2A.

14. The vector of claim 12, wherein at least one of the TEEs is resistant to the activity of the product which blocks host protein synthesis.

15. The vector of claim 1, wherein the two or more transcriptional elements are selected from the group consisting of minimal promoters, regulatable promoters, upstream activating sequences, and bacteriophage RNA polymerase specific promoters.

16. The vector of claim 1, wherein the first cistron encodes GAL4/VP16, GAL4, or a bacteriophage specific RNA polymerase.

17. The vector of claim 1, wherein at least one of the TEEs is an IRES, HCV-IRES or IRES element.

18. The vector of claim 1, wherein the IRES, HCV-IRES or IRES element is a Gtx sequence.

19. The vector of claim 1, wherein at least one of the TEEs is an N18 sequence, wherein N18 is a random oligonucleotide.

20. A nucleic acid vector comprising a first construct comprising a first transcriptional element, a first cistron encoding a first gene product, and a first translational enhancer element (TEE), wherein the TEE is resistant to an activity of a second gene product which blocks host protein synthesis.

21. The vector of claim 20, wherein the vector comprises one or more second constructs which encode the second gene product.

22. The vector of claim 20, wherein the TEE is an IRES, HCV-IRES or IRES element.

23. The vector of claim 22, wherein the IRES, HCV-IRES or IRES element is a Gtx sequence.

24. The vector of claim 20, wherein at least one of the TEEs is an N18 sequence, wherein N18 is a random oligonucleotide.

25. The vector of claim 20, wherein the second gene product is NSP3, L-proteinase, or proteinase 2A.

26. A method of identifying a translational enhancer element (TEE) comprising:

a) inserting nucleotides from a library of nucleotides into a vector comprising a first construct comprising two or more first transcriptional elements, and a first cistron encoding a transcription factor;

b) transfecting a cell with the vector of step (a);

and

c) determining the level of gene product translation from one or more second constructs in the cell of step (b),

wherein determining an enhanced level of translation of the gene product in the presence of the inserted nucleotides is indicative of the presence of at least one TEE.

27. The method of claim 26, further comprising co-transfecting the cell of step (b) with one or more second vectors comprising the one or more second constructs.

28. The method of claim 26, wherein at least one of the sequences comprising the first transcriptional elements is not the target of transcription factors endogenous to the cell.

29. The method of claim 26, wherein the transcription factor is not endogenous to the cell.

30. The method of claim 26, wherein two or more second constructs comprise at least one transcriptional element and a TEE which are 5′

-proximal to at least one cistron contained therein.

31. The method of claim 30, wherein one of the second constructs comprises a cistron encoding a gene product which blocks host protein synthesis.

32. The method of claim 31, wherein the blocking gene product is NSP3, L-proteinase, or proteinase 2A.

33. The method of claim 31, wherein the at least one TEE is an IRES, HCV-IRES or IRES element.

34. The method of claim 33, wherein the IRES, HCV-IRES or IRES element is a Gtx sequence.

35. The method of claim 33, wherein at least one TEE is resistant to the activity of the product which blocks host protein synthesis.

36. The method of claim 26, wherein the two or more transcriptional elements are selected from the group consisting of minimal promoters, regulatable promoters, upstream activating sequences, and bacteriophage RNA polymerase specific promoters.

37. The method of claim 26, wherein the first cistron encodes GAL4/VP16, GAL4, or a bacteriophage specific RNA polymerase.

45. The method of claim 33, wherein the at least one TEE is an IRES, HCV-IRES or IRES element.

46. The method of claim 45, wherein the IRES, HCV-IRES or IRES element is a Gtx sequence.

47. The method of claim 33, wherein the at least one TEE is an N18 sequence, wherein N18 is a random oligonucleotide.

38. A method of overexpressing a gene comprising:

a) transfecting a cell with a vector comprising a first construct comprising two or more first transcriptional elements, a first cistron encoding a transcription factor, and one or more first translation enhancer elements (TEEs); and

b) expressing, in the cell of step (a), a gene product from one or more second constructs comprising a second TEE,

wherein the resulting level of gene product expressed from the second construct is enhanced in the presence of the first and second TEEs.

39. The method of claim 38, further comprising co-transfecting the cell of step (a) with one or more second vectors comprising the one or more second constructs.

40. The method of claim 38, further comprising expressing, in the cell of step (a), a gene from a third construct comprising a third TEE, wherein the gene from the third construct encodes one or more gene products which block host protein synthesis.

41. The method of claim 40, further comprising co-transfecting the cell of step (a) with a third vector comprising the third construct.

42. The method of claim 40, wherein the blocking gene product is NSP3, L-proteinase, or proteinase 2A.

43. The method of claim 38, wherein the two or more transcriptional elements are selected from the group consisting of minimal promoters, regulatable promoters, upstream activating sequences, and bacteriophage RNA polymerase specific promoters.

44. The method of claim 38, wherein the first cistron encodes GAL4/VP16, GAL4, or a bacteriophage specific RNA polymerase.

48. A method of overexpressing a gene comprising:

a) transfecting a cell with a vector comprising a first construct comprising at least one first transcriptional element, a first cistron encoding a first gene product, and at least one translational enhancer element (TEE); and

b) expressing, in the cell of step (a), a gene from one or more second constructs encoding a protein which blocks host protein synthesis with one or more TEEs,

wherein the resulting level of gene product expressed from the first construct is enhanced in the presence of the blocking protein.

49. The method of claim 48, further comprising co-transfecting the cell of step (a) with a second vector comprising the one or more second constructs.

50. The method of claim 48, wherein the TEE is resistant to the blocking protein.

51. The method of claim 48, wherein the two or more transcriptional elements are selected from the group consisting of minimal promoters and regulatable promoters.

52. The method of claim 48, wherein the at least one TEE is an IRES, HCV-IRES or IRES element.

53. The method of claim 52, wherein the IRES, HCV-IRES or IRES element is a Gtx sequence.

54. The method of claim 48, wherein the at least one TEE is an N18 sequence, wherein N18 is a random oligonucleotide.

55. The method of claim 48, wherein the blocking gene product is NSP3, L-proteinase, or proteinase 2A.