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

Amitabha De Amitabha, B. D. Paul, V. Ramesh, V. Nagaraja

Research output: Contribution to journalArticle

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.

Original languageEnglish (US)
Pages (from-to)935-941
Number of pages7
JournalProtein Engineering
Volume10
Issue number8
StatePublished - Nov 28 1997
Externally publishedYes

Keywords

  • DNA binding protein
  • Dimerization
  • Mu C
  • Protein A gene fusion

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology

Fingerprint Dive into the research topics of 'Use of protein A gene fusions for the analysis of structure-function relationship of the transactivator protein C of bacteriophage Mu'. Together they form a unique fingerprint.

  • Cite this