Activation induced deaminase (AID)
View Patent ↗The invention is directed to a cell comprising a nucleic acid encoding an Activation Induced Deaminase (AID) polypeptide, a fusion protein comprising an AID polypeptide, and methods of using a nucleic acid encoding an AID polypeptide.
1. An in vitro method for preparing a mutant immunoglobulin, which method comprises expressing in vitro in a eukaryotic cell a transgene comprising a nucleic acid encoding an immunoglobulin having a functional activity, wherein (i) the eukaryotic cell expresses AID mutation activity, (ii) expression of AID mutation activity in the eukaryotic cell induces a mutation in the nucleic acid encoding the immunoglobulin, and (iii) expression of the mutated nucleic acid produces a mutant immunoglobulin having a modulated functional activity as compared to the immunoglobulin prior to mutation.
2. The method of claim 1 , wherein the transgene comprising the nucleic acid encoding the immunoglobulin is in an expression vector.
3. The method of claim 2 , wherein the expression vector comprises an inducible promoter.
4. The method of claim 1 , wherein the transgene is chromosomally integrated into the genome of the eukaryotic cell.
5. The method of claim 4 , wherein the transgene became chromosomally integrated into the genome of the eukaryotic cell by homologous recombination.
6. The method of claim 2 , wherein the expression vector further comprises control sequences which direct hypermutation in the nucleic acid encoding the immunoglobulin.
7. The method of claim 1 , wherein AID mutation activity is mediated by an AID homolog selected from the group consisting of Apobec-1, Apobec3C, and Apobec3G.
8. The method of claim 1 , which is successively repeated.
9. The method of claim 1 , wherein AID mutation activity is transiently expressed in the eukaryotic cell.
10. The method of claim 1 , wherein AID mutation activity is stably expressed in the eukaryotic cell.
11. The method of claim 1 , which further comprises using binding to magnetic beads to determine whether the eukaryotic cell produces a mutant immunoglobulin having a modulated functional activity as compared to the immunoglobulin prior to mutation.
12. The method of claim 1 , which further comprises using affinity chromatography to determine whether the eukaryotic cell produces a mutant immunoglobulin having a modulated functional activity as compared to the immunoglobulin prior to mutation.
13. The method of claim 1 , wherein the functional activity is binding affinity for a ligand.
14. The method of claim 13 , wherein the ligand is an antigen.