We have designed a two-phagemid system for the construction of very large non-phage displayed Fab antibody libraries in E. coli approaching 1012 members. The system can accommodate both periplasmic and cytoplasmic Fab expression and should prove useful for the direct selection of functional antibodies by genetic techniques. We successfully alleviate problems of Fab vector instability and report a set of improved 5' primers for the amplification of mouse Ig V(H) repertoires from mouse spleen. These primers have no more than one mismatch in the last 11 bases for >95% of mouse Ig V(H) genes and minimize the amount of N-terminal amino acid changes while maintaining the flexibility of periplasmic or cytoplasmic antibody expression in E. coli. Copyright (C) 2000 Elsevier Science B.V.
All Science Journal Classification (ASJC) codes
- Immunology and Allergy