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Nucleic Acids Research, 1983, Vol. 11, No. 12 4077-4092
© 1983

MOLECULAR BIOLOGY

Solubilization and immune-detection of ß-galactosidase hybrid proteins carrying foreign antigenic determinants

Keith K. Stanley

European Molecular Biology Laboratory Meyerhofstrasse 1, Postfach 10.2209, 6900 Heidelberg, FRG

Received March 9, 1983. Revised May 12, 1983. Accepted May 20, 1983.

Three DNA fragments representing almost the entire E1 gene of Semliki Forest virus (SFV) were inserted into a cro-lacZ expression vector by oligo dC.oligo dG tailing. Fragments inserted close to the 5' end of the lacZ gene gave rise to hybrid proteins which were rapidly degraded. Insertion of the same fragments at the 3' end, however, resulted in the synthesis of stable hybrid proteins which precipitated in an insoluble form within the bacteria. Insufficient hybrid protein was soluble to allow detection by immunoradiometric assay. Colonies grown on nitrocellulose filters, however, could be solubilized in SDS and subsequently renatured such that antibodies raised against the intact or SDS-denatured El protein cross-reacted with the hybrid proteins in a high percentage of colonies. This model system demonstrates a simple procedure for identifying DNA exon fragments by the immunological detection of expressed hybrid proteins.


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