We have developed efficient methodologies for construction and expression of comprehensive phage display libraries of murine Fab antibody fragments in E.coli cells. Our methods optimize several critical steps of the polymerase chain reaction (PCR) amplification of transcripts of the re-arranged immunoglobulin genes and of their subsequent assembly and expression: Firstly, we have designed exhaustive sets of PCR primers of low degeneracy for the amplification of transcripts of the Fab region of the heavy and lightchain genes. These primers proved effective in amplification of Fab gene fragments from a large panel of hybridoma cell lines of different specificity and family sub-type. Secondly, we have developed a 'jumping PCR' technique that effectively assembled and recombined the amplified heavy and light-chain gene fragments into a bi-cistronic operon. Thirdly, we have constructed expression vectors for insertion of the combinatorial Fab gene-cassette in fusion with a truncated version of the phage surface protein, glllp. The heavy chain and the light chain-gill fusion are transcribed as a polycistronic mRNA from the laczpromoter and efficient transcriptional control is provided by wildtype lacl present on the vector. The utility of the system was demonstrated by isolating several antigen-binding clones from hybridomas and libraries made from immunized mice.
展开▼
