A high-stringency proteomics concept aimed for generation of antibodies specific for cDNA-encoded proteins.

Abstract

A novel dual bacterial expression system, designed for high-throughput generation of antibodies specific for cDNA-encoded proteins, is presented. The concept involves parallel expression of cDNA-encoded proteins, in two vector systems, as fusions with two different tags, both enabling single-step affinity purification under denaturing conditions. One of the fusion tags includes a portion with documented immunopotentiating effect to stimulate antibody production, and the generated fusion proteins are used to elicit antibodies. The second fusion protein is used in an immobilized form as an affinity ligand to enrich, from the generated antisera, antibodies with selective reactivity to the cDNA-encoded part. To evaluate the system, five cDNA clones from a mouse testis cDNA library were expressed, and antibodies to these cDNA-encoded proteins were generated, enriched and used in blotting procedures to determine expression patterns for the native proteins corresponding to the cDNAs. The five antibody preparations showed specific reactivity to the corresponding recombinant cDNA-encoded proteins, and three of the five antibodies gave specific staining in Western-blot screening of various cell types and tissue homogenates. When the same five cDNAs were processed and analysed using a single-vector method, antibodies with a more non-specific staining were generated. We thus conclude that the presented dual-vector method offers a highly stringent strategy for generation of monospecific polyclonal antibodies.