Activation Induced Deaminase (AID)
View Patent ↗The present invention identifies that the expression of Activation Induced Deaminase (AID) or its homologues in cells confers a mutator phenotype and thus provides a method for generating diversity in a gene or gene product as well as cell lines capable of generating diversity in defined gene products. The invention also provides methods of modulating a mutator phenotype by modulating AID expression or activity.
1. A method for preparing a mutant immunoglobulin, which method comprises:
expressing in a prokaryotic cell a transgene comprising a nucleic acid encoding an immunoglobulin having a functional activity, wherein
(i) the prokaryotic cell expresses AID mutation activity,
(ii) expression of AID mutation activity in the prokaryotic 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 nucleic acid encoding the immunoglobulin is operably linked to the second half of a specific binding pair.
3. The method of claim 1 , wherein said transgene is expressed via an expression vector comprising said nucleic acid encoding said immunoglobulin.
4. The method of claim 3 , wherein said expression vector comprises an inducible promoter.
5. The method of claim 1 , wherein said transgene has undergone chromosomal integration into the genome of the prokaryotic cell.
6. The method of claim 5 , wherein said chromosomal integration is via homologous recombination.
7. The method of claim 3 , wherein said expression vector further comprises control sequences which direct hypermutation in said nucleic acid encoding said immunoglobulin.
8. The method of claim 1 , wherein the genetic background of the prokaryotic cell confers a UDG deficiency on the prokaryotic cell.
9. The method of claim 1 , wherein AID mutation activity is mediated by a homologue selected from Apobec-1, Apobec3C or Apobec3G.
10. The method of claim 1 , which is successively repeated.
11. The method of claim 1 , wherein AID mutation activity is transiently expressed.
12. The method of claim 1 , wherein AID mutation activity is stably expressed.
13. The method of claim 1 , wherein said prokaryotic cell is a bacterial cell.
14. The method of claim 13 , wherein said bacterial cell is bacteria E. coli.
15. The method of claim 1 , which further comprises maintaining the prokaryotic cell in a selectable medium to determine whether the prokaryotic cell produces a mutant immunoglobulin having a modulated functional activity as compared to the immunoglobulin prior to mutation.
16. The method of claim 1 , which further comprises using binding to magnetic beads to determine whether the prokaryotic cell produces a mutant immunoglobulin having a modulated functional activity as compared to the immunoglobulin prior to mutation.
17. The method of claim 1 , which further comprises using affinity chromatography to determine whether the prokaryotic cell produces a mutant immunoglobulin having a modulated functional activity as compared to the immunoglobulin prior to mutation.
18. The method of claim 1 , wherein the functional activity is binding affinity for a ligand.
19. The method of claim 18 , wherein the ligand is an antigen.
20. The method of claim 1 , wherein the functional activity is enzymatic activity.
21. The method of claim 1 , wherein the prokaryotic cell is modified to express a fusion protein comprising AID activity operably linked to one half of a specific binding pair having a mutator phenotype.
22. The method of claim 21 , wherein the specific binding pair is a DNA binding domain.