Co-expression of antibody fab heavy and light chain genes from separate evolved compatible replicons in E. coli.
Antibody molecules bind to antigen with six complementary determining region (CDR) loops, three of which are located on each variable heavy (V(H)) and light (V(L)) chains. Discovery and optimization of antibodies that bind antigen using in vitro techniques require diversification of one or more of these CDRs. Since antibodies are dimeric, simultaneous diversification of heavy and light chains on separate genetic elements would allow "chain shuffling" to occur simply and efficiently. Efficient expression of antibody V(H) and V(L) requires that the two separate replicons be compatible with one another, but also have similar properties, such as copy number in E. coli. Standard plasmids that are compatible with one another in E. coli exist at widely variable copy numbers. Recently we described the isolation of ColE1 mutants that have similar copy numbers but different incompatibility characteristics. Thus, new compatibility groups in the ColE1 family were established. Herein we describe the E. coli expression of V(H) and V(L) genes to form a functional Fab. The ability to express antibody heavy and light chains from separate but compatible high copy plasmids should allow new opportunities in antibody engineering, such as rapid chain shuffling and generation of more complex antibody libraries.
Leonard
Brandon
B
IntegriGen, Inc., 42 Digital Dr. Suite 6, Novato, CA 94949, USA.
Sharma
Vikram
V
Smider
Vaughn
V
D005075:Biological Evolution; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000235:genetics* / Q000276:immunology; D050438:Genes, Immunoglobulin Heavy Chain; D050439:Genes, Immunoglobulin Light Chain; D015151:Immunoblotting; D007140:Immunoglobulin Fab Fragments / Q000235:genetics*; D010957:Plasmids / Q000235:genetics*; D012093:Replicon / Q000235:genetics*; D014169:Transformation, Bacterial
D007140:Immunoglobulin Fab Fragments