Detection and use of recombinant staphylococcal protein A fusion proteins to localize nucleic-acid-binding domains of proteins.
We developed a rapid method to visualize recombinant staphylococcal protein A fused to segments of another protein and detect DNA-binding proteins simultaneously. Fusion genes were expressed in Escherichia coli to produce fusion proteins of protein A and segments of c-myc oncoprotein. Polypeptides from total E. coli lysates were separated in sodium dodecyl sulfate-polyacrylamide gels and transferred onto nitrocellulose membranes. Without the use of an antigen-specific antibody, protein A/c-myc fusion proteins were visualized by incubation of nitrocellulose membranes with horseradish-peroxidase (HRP)-conjugated immunoglobulin, the Fc region of which directly binds protein A. The sensitivity of detection of the protein A/c-myc fusion proteins was greatly enhanced by incubation of nitrocellulose membranes with rabbit immunoglobulin before incubation with HRP-conjugated goat anti-rabbit antibody. Prior to incubation of nitrocellulose membranes with immunoglobulin, the membranes were incubated with radiolabeled DNA to visualize DNA-binding proteins by autoradiography. Purified staphylococcal protein A does not bind DNA, whereas a segment of c-myc oncoprotein fused to protein A binds DNA in vitro. These methods may be applied to identify nucleic-acid-binding domains of other proteins without prior purification of the fusion proteins.
Dang
C V
CV
Department of Medicine, Johns Hopkins University, School of Medicine, Baltimore, Maryland 21205.
D001665:Binding Sites; D003001:Cloning, Molecular; D003101:Collodion; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D015151:Immunoblotting; D011518:Proto-Oncogene Proteins / Q000235:genetics / Q000378:metabolism; D016271:Proto-Oncogene Proteins c-myc; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism*; D013205:Staphylococcal Protein A / Q000235:genetics / Q000378:metabolism*
D004268:DNA-Binding Proteins; D011518:Proto-Oncogene Proteins; D016271:Proto-Oncogene Proteins c-myc; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D013205:Staphylococcal Protein A; D003101:Collodion