USE OF SOMATIC HYPERMUTATION TO CREATE INSERTION AND DELETION MUTATIONS IN VITRO
The invention is directed to an in vitro method for introducing one or more insertion mutations and/or one or more deletion mutations in a nucleic acid sequence encoding a polypeptide. The method comprises contacting a cell in vitro with a nucleic acid sequence, and expressing Activation Induced Cytidine Deaminase (AID) in the cell. The invention also is directed to a method of producing an immunoglobulin.
1 . A method for introducing one or more insertion mutations and/or one or more deletion mutations in a nucleic acid sequence encoding a polypeptide, which method comprises:
(a) providing a cell in vitro that expresses or can be induced to express Activation Induced Cytidine Deaminase (AID),
(b) contacting the cell in vitro with a nucleic acid sequence that encodes a polypeptide, and
(c) expressing AID in the cell, whereupon one or more insertion mutations and/or one or more deletion mutations are introduced in the nucleic acid sequence encoding the polypeptide.
2 . The method of claim 1 , wherein the nucleic acid sequence encodes an immunoglobulin heavy chain polypeptide.
3 . The method of claim 2 , wherein one or more insertion mutations are introduced in the nucleic acid sequence encoding the immunoglobulin heavy chain polypeptide.
4 . The method of claim 3 , wherein the one or more insertion mutations result in the insertion of one to eight amino acid residues into the immunoglobulin heavy chain polypeptide.
5 . The method of claim 2 , wherein one or more deletion mutations are introduced in the nucleic acid sequence encoding the immunoglobulin heavy chain polypeptide.
6 . The method of claim 5 , wherein the one or more deletion mutations result in the deletion of one to eight amino acid residues from the immunoglobulin heavy chain polypeptide.
7 . The method of claim 2 , wherein the one or more insertion mutations and/or the one or more deletion mutations occur in a complementarity determining region (CDR) of the immunoglobulin heavy chain polypeptide.
8 . The method of claim 7 , wherein the one or more insertion mutations and/or the one or more deletion mutations occur in CDR3 of the immunoglobulin heavy chain polypeptide.
9 . An isolated or purified nucleic acid sequence which encodes an immunoglobulin heavy chain polypeptide prepared by the method of claim 2 .
10 . The method of claim 1 , wherein the nucleic acid sequence encodes an immunoglobulin light chain polypeptide.
11 . The method of claim 10 , wherein one or more insertion mutations are introduced in the nucleic acid sequence encoding the immunoglobulin light chain polypeptide.
12 . The method of claim 11 , wherein the one or more insertion mutations result in the insertion of one to eight amino acid residues into the immunoglobulin light chain polypeptide.
13 . The method of claim 10 , wherein one or more deletion mutations are introduced in the nucleic acid sequence encoding the immunoglobulin light chain polypeptide.
14 . The method of claim 13 , wherein the one or more deletion mutations result in the deletion of one to eight amino acid residues from the immunoglobulin light chain polypeptide.
15 . The method of claim 10 , wherein the one or more insertion mutations and/or the one or more deletion mutations occurs in a complementarity determining region (CDR) of the immunoglobulin light chain polypeptide.
16 . The method of claim 15 , wherein the one or more insertion mutations and/or the one more deletion mutations occur in CDR3 of the immunoglobulin light chain polypeptide.
17 . An isolated or purified nucleic acid sequence which encodes an immunoglobulin light chain polypeptide prepared by the method of claim 10 .
18 . A method of producing an immunoglobulin, which method comprises:
(a) providing a cell in vitro that expresses or can be induced to express AID,
(b) contacting the cell in vitro with the nucleic acid sequence encoding an immunoglobulin heavy chain polypeptide of claim 9 and the nucleic acid sequence encoding an immunoglobulin light chain polypeptide of claim 17 , and
(c) expressing AID in the cell, whereupon an immunoglobulin comprising a heavy chain polypeptide and a light chain polypeptide is produced in the cell.
19 . The method of claim 18 , wherein the cell is a eukaryotic cell.
20 . The method of claim 19 , wherein the cell is a B-cell.
21 . The method of claim 19 , wherein the cell is an HEK-293 cell.
22 . The method of claim 1 , wherein the cell is a eukaryotic cell.