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

摘要

A sensitive dimerization assay for DNA binding proteins has been developed using gene fusion technology. For this purpose, we have engineered a gene fusion using protein A gene of Staphylococcus aureus and C gene, the late gene transactivator of bacteriophage Mu, The C gene was fused 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 exhibits DNA binding activity as demonstrated by electrophoretic mobility shift 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 single intermediate complex comprising of a heterodimer of C and A-C fusion proteins was observed, Further, the protein A moiety in the fusion protein A-C does not contribute to DNA binding as demonstrated by proteolytic cleavage and circular dichroism (CD) analysis, The assay has also been applied to analyze the DNA binding domain of C protein by generating 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 this method is discussed. [References: 18]