Expression vectors based on RK2 and TOL plasmids

• US 6,258,565 B1
• Filed: 09/10/1999
• Issued: 07/10/2001
• Est. Priority Date: 08/29/1996
• Status: Expired due to Term

• Current Assignee: Vectron Biosolutions AS
• Original Assignee: Leiv Eiriksson Nyfotek AS
• Inventors: Blatny, Janet M., Karunakaran, Ponniah, Valla, Svein
• Primary Examiner(s): Schwartzman, Robert A.
• Assistant Examiner(s): SANDALS, WILLIAM O
• Application Number:
  US09/242,945
• Time in Patent Office: 669 Days
• Field of Search: 435/71.2, 435/252.1, 435/320.1, 435/440, 536/23.1, 536/24.1
• US Class Current: 435/71.2
• CPC Class Codes: C12N 15/68 Stabilisation of the vector
• CPC Class Codes: C12N 15/69 Increasing the copy number of the vector
• CPC Class Codes: C12N 15/70 Vectors or expression systems specially adapted for E. coli
• CPC Class Codes: C12N 15/74 Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
• CPC Class Codes: C12N 9/92 Glucose isomerase (5.3.1.5; 5.3.1.9; 5.3.1.18)

Abstract

The present invention provides an expression vector comprising an RK2 minimum replicon together with an expression cassette comprising the regulatory functions of a TOL plasmid, and, in particular, an expression vector comprising a RK2 minimum replicon together with a promoter Pm and/or Pu and a corresponding regulatory gene xylS and/or xylR as derived from a TOL plasmid. Such expression vectors may be used to express desired genes in a wide range of gram negative and gram positive bacterial hosts.

Claims

1. An expression vector comprising an RK2 minimum replicon, including a trfa sequence, together with an expression cassette comprising a promoter Pm and a xylS sequence from a TOL plasmid, and/or a promoter Pu and a xylR sequence from a TOL plasmid.

2. An expression vector as claimed in claim 1 wherein, in said RK2 minimum replicon, the trfa sequence is a copy-up (COP) mutant.

3. An expression vector as claimed in claim 1, wherein said RK2 minimum replicon carries mutations in the trifA sequence that are temperature-sensitive for replication.

4. An expression vector as claim in claim 1 wherein the trfa sequence is under control of a Pm and/or Pu promoter.

5. An expression vector as claimed in claim 1, comprising Pm and a xyls, xyls2tr6, or xylsarg41pro sequence.

6. An expression vector as claimed in claim 1, comprising a polylinker/lacZ′

region.

7. An expression vector as claimed in claim 1, comprising an RK2-derived OriT.

8. An expression vector as claimed in claim 1, further comprising one or more further regulatory and/or enhancer elements.

9. An expression vector as claimed in claim 1, comprising a plasmid-encoded stability region.

11. A host cell containing an expression vector as defined in claim 1.

12. A host cell as claimed in claim 11, wherein said host cell is selected from the group consisting of Escherichia sp., Salmonella, Klebsiella, Proteus, Yersinia, Azotobacter sp., Pseudomonas sp., Xanthomonas sp., Caulobacter sp., Acinetobacter sp., Aeromonas sp., Agrobacterium sp., Alcaligenes sp., Bordatella sp., Haemophilus Influenzae, Methylophilus methylotrophus, Rhizobium sp., Thiobacillus sp., and Clavibacter sp.

13. A method of expressing a desired gene within a host cell, comprising introducing into said cell an expression vector as defined in claim 1 containing said desired gene, and culturing said cell under conditions in which said desired gene is expressed.

14. A method of preparing a desired polypeptide product by culturing a host cell containing an expression vector as defined in claim 1 into which a desired gene encoding said desired polypeptide has been introduced, under conditions whereby said polypeptide is expressed, and recovering said desired polypeptide thus produced.

15. A method as claimed in claim 13 or 14, wherein said host cell is selected from the group consisting of Escherichia sp., Salmonella, Klebsiella, Proteus, Yersinia, Azotobacter sp., Pseudomonas sp., Xanthomonas sp., Caulobacter sp., Acinetobacter sp., Aeromonas sp., Agrobacterium sp., Alcaligenes sp., Bordatella sp., Haemophilus Influenzae, Methylophilus methylotrophus, Rhizobium sp., Thiobacillus sp., and Clavibacter sp.

10. An expression vector as claimed in 9, wherein said plasmid-encoded stability region comprises par loci.