PURIFICATION OF E-COLI-SYNTHESIZED PAN PROTEINS AND DEVELOPMENT OF A PAN-SPECIFIC MONOCLONAL-ANTIBODY

The helix-loop-helix (HLH) transcription factors, Pan-1 (E47) and Pan- 2 (E12), are produced by the mechanism of alternative transcript splic ing. Pan-1 and Pan-2 were expressed in Escherichia coli, and a purific ation scheme was developed. Purified Pan-2 was used to immunize Smith- Webster mice and a hybridoma was generated that produced a monoclonal antibody (Yae) that specifically recognized both native and denatured Pan-1 and Pan-2. Deletion mapping and sequence transfer studies have l ocalized the determinant recognized by the Yae antibody to the region 195-208 of Pan-2, This region is conserved in Pan-1 and Pan-2. The Yae antibody recognized in vitro-synthesized ITF-1, a third E2A (Pan) gen e product also produced by the mechanism of alternative RNA splicing, but did not recognize the related HLH proteins, ITF-2, REB alpha, or R EB beta. By Western blot assay of pancreatic acinar cells, the Yae ant ibody detected a single protein species of 72 kD that comigrated with in vitro-synthesized Pan-1 and Pan-2.