Making Antibodies in Bacteria

Phage-Displayed Library .................................................. 169 8.3.2.4 Preparation of Electrocompetent E. coli SS320 .............. 170 8.3.2.5 E. coli Electroporation and Phage Propagation ............... 1718.3.3 Selection of Antigen-Specific Antibodies ....................................... 172 8.3.3.1 Selection against Immobilized Antigen ........................... 172 8.3.3.2 Selection against Biotinylated Antigen ............................ 173 8.3.3.3 Analysis of Binding Clones by Phage ELISA................. 1738.3.3.3.1 Direct-Binding Phage ELISA ........................ 174 8.3.3.3.2 Single-Point Competitive Phage ELISA........ 175 8.3.3.3.3 Multipoint Competitive Phage ELISA........... 1758.3.3.4 DNA Sequencing .............................................................. 176 8.3.3.5 Protein Purification........................................................... 176References.............................................................................................................. 177The bacterium Escherichia coli is a well-characterized and extremely robust host for recombinant protein expression. Among other advantages, expression in E. coli permits efficient mutagenesis and DNA manipulation, rapid establishment andFIGURE 8.1 (Caption on facing page)FabFvFcgrowth of expression cultures, and in ideal cases, high yields of purified proteins. Thus, the ability to express and purify antibody fragments from E. coli has been of great utility for both basic research into antibody structure and function and for the development of antibody reagents and therapeutics. However, because of the complex nature of the immunoglobulin molecule, the development of efficient expression protocols in bacteria faced significant challenges, which have nonetheless been gradually surmounted through the efforts of many researchers.