Method of cloning nucleic acid

Patent metadata

Field	Value
Publication number	US-9617329-B2
Application number	US-201113877213-A
Country	US
Kind code	B2
Filing date	Oct 3, 2011
Priority date	Oct 1, 2010
Publication date	Apr 11, 2017
Grant date	Apr 11, 2017

Abstract

The present disclosure provides methods for producing expression constructs comprising linking a plurality of unlinked nucleic acids, including a nucleic acid encoding a marker protein.

First claim

The invention claimed is:

1. A method for producing an expression construct capable of expressing a plurality of polypeptides, the method comprising:
providing the following unlinked nucleic acids:
(i) a first nucleic acid encoding a first polypeptide;
(ii) a second nucleic acid encoding a second polypeptide; and
(iii) a third nucleic acid encoding a marker protein that facilitates detection and/or isolation of a cell expressing the marker protein;
providing an expression vector, which does not comprise a nucleic acid encoding the marker protein encoded by the nucleic acid at (iii); and
linking said unlinked nucleic acids and said expression vector in a single cloning cycle, wherein said linking comprises ligase dependent cloning or ligase independent cloning, and wherein said first polypeptide and said second polypeptide associate to form a multimeric protein.

2. The method of claim 1 , wherein the nucleic acids are linked together using ligase independent cloning.

3. The method of claim 1 , wherein the expression vector comprises a nucleic acid encoding a further polypeptide that is expressed as a fusion with at least one of the polypeptides.

4. The method of claim 1 , wherein the nucleic acid(s) encoding the polypeptide(s) are each operably linked to a promoter.

5. The method of claim 4 , wherein the nucleic acid encoding the marker protein further comprises a promoter that becomes operably linked to at least one of the nucleic acids encoding a polypeptide.

6. The method of claim 1 , wherein the marker protein confers resistance to a toxic compound on a cell in which it is expressed or is a fluorescent protein or is a protein that metabolizes a substrate to produce a detectable compound or is a protein that confers a growth advantage on an auxotrophic cell.

7. The method of claim 1 , wherein the polypeptides each comprise an antibody variable region.

8. The method of claim 7 comprising linking the following unlinked nucleic acids:
(i) a nucleic acid encoding an antibody heavy chain variable region (V H );
(ii) a nucleic acid encoding a marker protein that facilitates detection and/or isolation of a cells expressing the marker protein and a promoter; and
(iii) an expression vector comprising a sequence encoding one or more antibody heavy chain constant regions, wherein the vector does not comprise a nucleic acid encoding the marker protein encoded by the nucleic acid at (ii);
such that:
(a) the promoter and the nucleic acid encoding the V H are operably linked; and
(b) the nucleic acid encoding the V H and the sequence encoding one or more antibody heavy chain constant regions are linked so as to encode a functional fusion protein.

9. The method of claim 7 comprising linking the following unlinked nucleic acids:
(i) a nucleic acid encoding an antibody light chain variable region (V L ) and, optionally a sequence encoding a light chain constant region positioned 3′ to the sequence encoding the V L ;
(ii) a nucleic acid encoding a marker protein that facilitates detection and/or isolation of a cells expressing the marker protein; and
(iii) an expression vector comprising a promoter, wherein the vector does not comprise a nucleic acid encoding the marker protein encoded by the nucleic acid at (ii),
such that:
(a) the promoter and the nucleic acid encoding the V L are operably linked; and
(b) the nucleic acid encoding the marker protein is positioned between the nucleic acid encoding the V L and the expression vector.

10. The method of claim 9 , wherein the nucleic acid encoding the marker protein comprises a polyadenylation signal, which following linking of the nucleic acids is operably linked to the nucleic acid encoding the V L .

11. The method of claim 7 comprising linking the following unlinked nucleic acids:
(i) a first nucleic acid encoding an antibody light chain variable region and a light chain constant region;
(ii) a nucleic acid encoding a marker protein that facilitates detection and/or isolation of a cells expressing the marker protein and a first promoter;
(iii) a second nucleic acid encoding an antibody heavy chain variable region; and
(iv) an expression vector comprising a sequence encoding one or more antibody heavy chain constant regions and a second promoter, wherein the vector does not comprise a nucleic acid encoding the marker protein encoded by the nucleic acid at (ii);
such that:
(a) the second promoter and first nucleic acid are operably linked;
(b) the nucleic acid encoding the marker protein is positioned between the first and second nucleic acids such that the first promoter is operably linked to the second nucleic acid;
(c) the second nucleic acid and said sequence encoding one or more antibody heavy chain constant regions are linked so as to encode a functional fusion protein.

12. The method of claim 1 , wherein the first polypeptide comprises an antibody light chain variable region (V L ) and, optionally an antibody light chain constant region and the second polypeptide comprises an antibody heavy chain variable region (V H ).

13. The method of claim 1 , further comprising performing the method a plurality of times in parallel, to thereby produce a plurality of expression constructs.

14. A method for producing a nucleic acid construct capable of expressing a plurality of polypeptides comprising:
providing the following unlinked nucleic acids:
(i) a first nucleic acid encoding a first polypeptide;
(ii) a second nucleic acid encoding a marker protein that facilitates detection and/or isolation of a cell expressing the marker protein; and
(iii) a third nucleic acid encoding a second polypeptide; and
linking said unlinked nucleic acids in a single cloning cycle, such that the second nucleic acid encoding the marker protein is positioned between the first and third nucleic acids, wherein said linking comprises ligase dependent cloning or ligase independent cloning, wherein said linking of said first, said second and said third nucleic acid forms said nucleic acid construct, and wherein said first polypeptide and said second polypeptide associate to form a multimeric protein.

15. The method of claim 14 , further comprising inserting the linked nucleic acids into an expression vector.

16. A process for producing a cell expressing a multimeric protein comprising:
(i) producing an expression construct in accordance with the method set forth in claim 1 ;
(ii) introducing the expression construct into a cell; and
(iii) selecting a cell comprising the expression construct.

17. The process of claim 16 , wherein the marker protein of step (i) confers resistance to a toxic compound on a cell in which it is expressed, and said selecting a cell comprising the expression construct comprises exposing the cell to the toxic compound.

18. The process of claim 16 , wherein each of steps (i), (ii) and (iii) are performed in solution in a single reaction vessel.

19. The process of claim 18 , wherein each of steps (i), (ii) and (iii) are performed without culturing cells on a solid medium.

20. A process for producing a multimeric protein comprising:
(i) producing an expression construct in accordance with the method set forth in claim 1 ;
(ii) maintaining the expression construct under conditions such that the multimeric protein is expressed to thereby produce the multimeric protein; and
(iii) optionally, purifying the multimeric protein.

21. The process of claim 20 , wherein the multimeric protein comprises an antibody variable

Assignees

• Panousis Con
• Chen Chao-Guang
• Csl Ltd

Inventors

• Panousis Con
• Chen Chao-Guang

Classifications

• G01N33/563

  involving antibody fragments · CPC title

• C12N15/65 Primary

  using markers (enzymes used as markers C12N15/52) · CPC title

• C07K16/00 Primary

  Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies · CPC title