Open AccessSolubilization and immune-detection of β-galactosidase hybrid proteins carrying foreign antigenic determinants
Author(s) -
Keith K. Stanley
Publication year - 1983
Publication title -
nucleic acids research
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 9.008
H-Index - 537
eISSN - 1362-4954
pISSN - 0305-1048
DOI - 10.1093/nar/11.12.4077
Subject(s) - biology , semliki forest virus , nitrocellulose , lac operon , microbiology and biotechnology , gene , dna , antigen , two hybrid screening , biochemistry , gene expression , genetics , membrane , rna
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 E1 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.