NOVEL VECTOR FOR GENE EXPRESSION IN PROKARYOTIC AND EUKARYOTIC SYSTEMS

• US 20010016351A1
• Filed: 10/31/1997
• Published: 08/23/2001
• Est. Priority Date: 10/31/1997
• Status: Active Grant

• Current Assignee: Stratagene California (Agilent Technologies, Inc.)
• Original Assignee: Stratagene California (Agilent Technologies, Inc.)
• Inventors: SORGE, JOSEPH A., PADGETT, KERSTIEN A.
• Granted Patent: US 6,627,436 B2
• Time in Patent Office: Days
• US Class Current: 435/320.1
• CPC Class Codes: C12N 15/72 Expression systems using regulatory sequences derived from the lac-operon
• CPC Class Codes: C12N 15/85 for animal cells
• CPC Class Codes: C12N 2800/108 episomal vectors
• CPC Class Codes: C12N 2830/002 inducible enhancer/promoter combination, e.g. hypoxia, iron, transcription factor
• CPC Class Codes: C12N 2830/15 chimeric enhancer/promoter combination
• CPC Class Codes: C12N 2830/55 from bacteria
• CPC Class Codes: C12N 2840/20 translation of more than one cistron
• CPC Class Codes: C12N 2840/44 being a specific part of the splice mechanism, e.g. donor, acceptor

Abstract

The invention concerns an expression vector that permits expression of genes and fragments thereof in both prokaryotic and mammalian systems. The invention also pertains to derivatives of such vector that contain one or more prokaryotic or eukaryotic (especially mammalian) genes or gene fragments. The invention further pertains to prokaryotic or mammalian cells containing such an expression vector or derivative.

Claims

1. A dual expression vector, said vector comprising:

(A) a cloning site;

(B) transcription elements sufficient to permit transcription of a polynucleotide inserted into said cloning site in both a prokaryotic and a eukaryotic cell;

(C) translation elements sufficient to permit translation of an RNA transcript of said polynucleotide in both a prokaryotic and a eukaryotic cell; and

(D) replication elements sufficient to permit the replication of said vector in;

wherein said vector mediates the high level expression of said inserted polynucleotide in both a prokaryotic and a eukaryotic cell.

2. The dual expression vector of

claim 1

, wherein said vector is a plasmid.

3. The dual expression vector of

claim 1

, wherein said cloning site is a restriction site.

4. The dual expression vector of

claim 1

, wherein said restriction site is a site cleaved by EcoRI restriction endonuclease.

5. The dual expression vector of

claim 3

, wherein said cloning site is flanked on each side by an Eam restriction site, said flanking Eam restriction sites being in inverted sequence orientation with respect to each other.

6. The dual expression vector of

claim 1

, wherein said transcription elements comprise a prokaryotic promoter that is inducible.

7. The dual expression vector of

claim 6

, wherein said inducible prokaryotic promoter is selected from the group consisting of a T7/LacO hybrid promoter, a trp promoter, a T7 promoter, a lac promoter, and a bacteriophage lambda promoter.

8. The dual expression vector of

claim 7

, wherein said inducible prokaryotic promoter is a T7/LacO hybrid promoter.

9. The dual expression vector of

claim 8

, which additionally comprises an expressed lacI gene.

10. The dual expression vector of

claim 9

, wherein said lacI gene is a lacIq allele of E. coli lacI.

11. The dual expression vector of

claim 8

, wherein said vector additionally comprises a T7 polymerase termination sequence.

12. The dual expression vector of

claim 1

, wherein said transcription elements comprise a eukaryotic promoter selected from the group consisting of a metallothionein promoter, an SV40 promoter, a retroviral promoter, a cytomegalovirus promoter, a tissue-specific promoter, and a tumor-specific promoter.

13. The dual expression vector of

claim 12

, wherein said eukaryotic promoter is a cytomegalovirus promoter.

14. The dual expression vector of

claim 13

, wherein said cytomegalovirus promoter is the cytomegalovirus promoter of pdual.

15. The dual expression vector of

claim 12

, wherein said transcription elements additionally comprise a polyadenylation element sufficient to polyadenylate the terminus of an RNA transcript of said inserted polynucleotide in a eukaryotic cell.

16. The dual expression vector of

claim 14

, wherein said transcription elements additionally comprise a polyadenylation element sufficient to polyadenylate the terminus of an RNA transcript of said inserted polynucleotide in a eukaryotic cell.

17. The dual expression vector of

claim 1

, wherein said translation elements comprise a polynucleotide encoding a tag polypeptide, such that said inserted polynucleotide is expressed as a fusion protein having a carboxy-terminal tag.

18. The dual expression vector of

claim 17

, wherein said tag polypeptide permits the affinity purification of said expressed polynucleotide.

19. The dual expression vector of

claim 18

, wherein said tag polypeptide is a calmodulin binding peptide.

20. The dual expression vector of

claim 17

, wherein said fusion protein contains a cleavable linkage preceding said tag polypeptide, such that said tag polypeptide may be separated from an expression product of said inserted polynucleotide by a cleaving agent.

21. The dual expression vector of

claim 20

, wherein said cleavable linkage is a thrombin-cleavable linkage, and wherein said cleaving agent is thrombin.

22. The dual expression vector of

claim 1

, wherein said replication elements are sufficient to permit the replication of said vector in E. coli.

23. The dual expression vector of

claim 22

, wherein said replication elements comprise a ColE1 origin of replication.

24. The dual expression vector of

claim 1

, wherein said replication elements are sufficient to permit the replication of said vector in human, murine, simian, bovine, porcine or ovine cells.

25. The dual expression vector of

claim 24

, wherein said replication elements are sufficient to permit the replication of said vector in human cells in culture.

26. The dual expression vector of

claim 24

, wherein said replication elements comprise an SV40 origin of replication.

27. The dual expression vector of

claim 1

, which additionally expresses a selectable marker.

28. The dual expression vector of

claim 27

, wherein said selectable marker is a gene that confers cellular resistance to an antibiotic.

29. The dual expression vector of

claim 28

, wherein said antibiotic is selected from the group consisting of G418 and neomycin.

30. The dual expression vector of

claim 1

, wherein said vector is pdual.

31. The dual expression vector of

claim 1

, wherein said vector additionally comprises a polynucleotide inserted into said cloning site.

32. The dual expression vector of

claim 31

, wherein said polynucleotide encodes a mammalian protein.

33. The dual expression vector of

claim 31

, wherein said mammalian protein is a human protein.

34. A process for obtaining high level expression of a protein encoded by a polynucleotide that comprises:

(A) inserting said polynucleotide into a vector that comprises;

(1) a cloning site;

(2) transcription elements sufficient to permit transcription of a polynucleotide inserted into said cloning site in both a prokaryotic and a eukaryotic cell;

(3) translation elements sufficient to permit translation of an RNA transcript of said polynucleotide in both a prokaryotic and a eukaryotic cell; and

(4) replication elements sufficient to permit the replication of said vector in both a prokaryotic and a eukaryotic cell;

(B) introducing said vector containing said inserted polynucleotide into a prokaryotic or a eukaryotic cell; and

(C) permitting said cell to mediate the high level expression of said inserted polynucleotide.

35. A kit for expressing a protein encoded polynucleotide that comprises:

(A) a first container containing a vector that comprises;

(1) a cloning site, comprising a restriction site, said cloning site being flanked on each side by an Eam restriction site, said flanking Eam restriction sites being in inverted sequence orientation with respect to each other;

(2) transcription elements sufficient to permit transcription of a polynucleotide inserted into said site in both a prokaryotic and a eukaryotic cell;

(3) translation elements sufficient to permit translation of an RNA transcript of said polynucleotide in both a prokaryotic and a eukaryotic cell; and

(4) replication elements sufficient to permit the replication of said vector in both a prokaryotic and a eukaryotic cell; and

(B) a second container containing Eam restriction endonuclease for cleaving said Eam restriction sites of said vector of said first container.