USE OF PROTEIN-A GENE FUSIONS FOR THE ANALYSIS OF STRUCTURE-FUNCTION RELATIONSHIP OF THE TRANSACTIVATOR PROTEIN-C OF BACTERIOPHAGE-MU

A sensitive dimerization assay for DNA binding proteins has been devel oped using gene fusion technology. For this purpose, we have engineere d a gene fusion using protein A gene of Staphylococcus aureus and C ge ne, the late gene transactivator of bacteriophage Mu, The C gene was f used to the 3' end of the gene for protein A to generate an A-C fusion . The overexpressed fusion protein was purified in a single step using immunoglobulin affinity chromatography. Purified fusion protein exhib its DNA binding activity as demonstrated by electrophoretic mobility s hift assays, When the fusion protein A-C was mixed with C and analyzed for DNA binding, in addition to C and A-C specific complexes, a singl e intermediate complex comprising of a heterodimer of C and A-C fusion proteins was observed, Further, the protein A moiety in the fusion pr otein A-C does not contribute to DNA binding as demonstrated by proteo lytic cleavage and circular dichroism (CD) analysis, The assay has als o been applied to analyze the DNA binding domain of C protein by gener ating fusions between protein A and N- and C-terminal deletion mutants of C, The results indicate a role for the region towards the carboxy terminal of the protein in DNA binding. The general applicability of t his method is discussed.