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 Nagaraja, V (1997) 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.

Preview

PDF 17_protegg.pdf Download (263kB)

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 39 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 Unversity Press
Keywords:	Dimerization;DNA binding protein;Mu C;protein A gene fusion
Department/Centre:	Division of Biological Sciences > Microbiology & Cell Biology
Date Deposited:	02 Sep 2004
Last Modified:	19 Sep 2010 04:13
URI:	http://eprints.iisc.ac.in/id/eprint/1023

Actions (login required)

View Item