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 splicing. Pan-1 and Pan-2 were expressed in Escherichia coli, and a purification 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 localized 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) gene product also produced by the mechanism of alternative RNA splicing, but did not recognize the related HLH proteins, ITF-2, REB alpha, or REB beta. By Western blot assay of pancreatic acinar cells, the Yae antibody detected a single protein species of 72 kD that comigrated with in vitro-synthesized Pan-1 and Pan-2.
Vierra
C A
CA
Department of Biochemistry, University of California, Riverside 92521.
Xin
X Q
XQ
Jacobs
Y T
YT
Campbell
J M
JM
Shen
L P
LP
Rutter
W J
WJ
Nelson
C
C
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology* / Q000302:isolation & purification; D001483:Base Sequence; D015153:Blotting, Western; D002460:Cell Line; D004268:DNA-Binding Proteins / Q000276:immunology* / Q000302:isolation & purification*; D000939:Epitopes / Q000276:immunology; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D018257:Helix-Loop-Helix Motifs; D051379:Mice; D008969:Molecular Sequence Data; D014176:Protein Biosynthesis / Q000235:genetics; D051381:Rats; D011994:Recombinant Proteins / Q000276:immunology / Q000302:isolation & purification; D051820:TCF Transcription Factors; D058125:Transcription Factor 7-Like 1 Protein; D014157:Transcription Factors / Q000276:immunology* / Q000302:isolation & purification*; D014158:Transcription, Genetic / Q000235:genetics
D000911:Antibodies, Monoclonal; D004268:DNA-Binding Proteins; D000939:Epitopes; D011994:Recombinant Proteins; D051820:TCF Transcription Factors; C544913:Tcf7l1 protein, mouse; D058125:Transcription Factor 7-Like 1 Protein; D014157:Transcription Factors