Rapid subcloning using site-specific recombination

• US 6,828,093 B1
• Filed: 07/24/1998
• Issued: 12/07/2004
• Est. Priority Date: 02/28/1997
• Status: Expired due to Fees

• Current Assignee: Baylor College of Medicine (Baylor College)
• Original Assignee: Baylor College of Medicine (Baylor College)
• Inventors: Liu, Qinghua, Elledge, Stephen J.
• Primary Examiner(s): KETTER, JAMES S
• Application Number:
  US09/122,384
• Time in Patent Office: 2,328 Days
• Field of Search: 435/91.4, 435/91.41, 435/320.1, 435/476, 435/477, 435/194, 435/6, 435/402, 435/252.33
• US Class Current: 435/6.18
• CPC Class Codes: C12N 15/10 Processes for the isolation, preparation or purification of DNA or RNA chemical preparation of DNA or RNA C07H21/00; preparation of non-structural polynucleotides from microorganisms or with enzymes C12P19/34
• CPC Class Codes: C12N 15/66 General methods for inserting a gene into a vector to form a recombinant vector using cleavage and ligation; Use of non-functional linkers or adaptors, e.g. linkers containing the sequence for a restriction endonuclease
• 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 15/905 in yeast

Abstract

The present invention provides compositions, including vectors, and methods for the rapid subcloning of nucleic acid sequences in vivo and in vitro. In particular, the invention provides vectors used to contain a gene of interest that comprise a sequence-specific recombinase target site. These vectors are used to rapidly transfer the gene or genes of interest into any vector that contains a sequence-specific recombinase target site located downstream of a regulatory element so that the gene of interest may be regulated.

Claims

1. A composition comprising a glutathione-S-transferase-Cre-recombinase fusion polypeptide.

2. The composition of claim 1, wherein the polypeptide has an amino acid sequence according to SEQ ID NO:

11.

3. The composition of claim 1, wherein the composition comprises an enzyme activity with a Cre recombinase efficiency of about 16.8% per microgram of protein.

4. An isolated nucleic acid molecule comprising a coding region wherein the coding region encodes a glutathione-S-transferase-Cre-recombinase fusion polypeptide.

5. The nucleic acid molecule of claim 4, wherein the coding region comprises the nucleic acid sequence of SEQ ID NO:

10.

6. The nucleic acid molecule of claim 4, wherein the isolated nucleic acid molecule is an expression vector.

7. The nucleic acid molecule of claim 4, wherein the coding region is operatively linked to a promoter effective to direct expression of a glutathione-S-transferase-Cre recombinase fusion polypeptide.

8. The nucleic acid molecule of claim 7, wherein the promoter is an inducible promoter.

9. The nucleic acid of claim 8, wherein the promoter is the tac promoter.

10. A host cell comprising the nucleic acid molecule of claim 4.

11. A host cell comprising the nucleic acid molecule of claim 7.

12. The host cell of claim 11, wherein the host cell expresses a Cre recombinase activity.

13. The host cell of claim 11, further defined as an E. coli cell.

14. A bacterial cell engineered to express a glutathione-S-transferase-Cre-recombinase fusion polypeptide.

15. The bacterial cell of claim 14, wherein the polypeptide has an amino acid sequence according to SEQ ID NO:

11.

16. A method of producing a glutathione-S-transferase-Cre-recombinase fusion polypeptide comprising:

obtaining an expression vector comprising a coding region encoding a glutathione-S-transferase-Cre-recombinase fusion polypeptide operatively linked to a promoter;

transforming or transfecting the vector into a cell; and

growing the cell under conditions effective to express a glutathione-S-transferase-Cre-recombinase fusion polypeptide.

17. The method of claim 16, further comprising isolating the glutathione-S-transferase-Cre-recombinase fusion polypeptide.

18. The method of claim 17, wherein isolating the polypeptide comprises glutathione affinity chromatography.

19. A method of recombining nucleic acid segments, wherein each segment comprises a lox site specific recombinase site, the method comprising contacting the nucleic acid segment with a glutathione-S-transferase-Cre-recombinase fusion polypeptide.

20. The method of claim 19, wherein the polypeptide has an amino acid sequence according to SEQ ID NO:

11.

21. A composition comprising a glutathione-S-transferase-Cre-recombinase fusion polypeptide and one or more nucleic acid molecules, wherein the nucleic acids comprise a site specific recombinase site.

22. The composition of claim 21, wherein at least one of said nucleic acid molecules comprises a lox recombination site upstream in a 5′

to 3′

orientation from an amino acid encoding region.

23. The composition of claim 21, wherein at least one of said nucleic acid molecules comprises a transcription regulatory element upstream in a 5′

to 3′

orientation of a lox recombinase site.

24. The composition of claim 22 wherein the lox recombinase site is a loxP, loxP2, loxP3, loxP23, loxP511, loxB, loxC2, loxL, loxR, loxΔ

86, loxΔ

117, or loxH site.

25. The composition of claim 23 wherein the lox recombinase site is a loxP, loxP2, loxP3, loxP23, loxP511, loxB, loxC2, loxL, loxR, loxΔ

86, loxΔ

117, or loxH site.

26. The composition of claim 22, wherein the amino acid encoding region is a member of a nucleic acid library.