From EST to IHC: human antibody pipeline for target research.
We have developed a method for the high-level expression of expressed sequence tags (ESTs) as inclusion bodies in Escherichia coli by C-terminal fusion to the N1-domain of g3p of filamentous phage M13. Soluble fusion protein is obtained by an efficient refolding procedure. We have applied such protein preparations to the selection of human antibody fragments from phage-displayed HuCAL libraries. For all fusion proteins tested in this study, HuCAL antibodies could be generated which specifically detect, e.g. in immunohistochemistry, the maternal full-length protein corresponding to the protein fragment. This expression technology, in combination with the automated HuCAL antibody generation (AutoCAL), has proven to be useful for the rapid, high-throughput generation of high-quality human antibodies against EST-encoded protein fragments for target research.