Use of protein A gene fusions for the analysis of structure-function relationship of the transactivator protein C of bacteriophage Mu

De, Amitabha and Paul, BD and Ramesh, V and Nagaraja, V (1977) Use of protein A gene fusions for the analysis of structure-function relationship of the transactivator protein C of bacteriophage Mu. In: Protein Engineering, 10 (8). pp. 935-941.

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Abstract

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.

Item Type:	Journal Article
Publication:	Protein Engineering
Publisher:	Oxford University Press
Additional Information:	Copyright for this article belongs to Oxford University Press.
Department/Centre:	Division of Biological Sciences > Molecular Reproduction, Development & Genetics
Date Deposited:	23 Jan 2010 09:37
Last Modified:	19 Sep 2010 05:51
URI:	http://eprints.iisc.ac.in/id/eprint/24699

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