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 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.
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Centre for Genetic Engineering, Indian Institute of Science, Bangalore.
Paul
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Ramesh
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Nagaraja
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D000595:Amino Acid Sequence; D019677:Artificial Gene Fusion; D051778:Basic Helix-Loop-Helix Leucine Zipper Transcription Factors; D001665:Binding Sites; D002853:Chromatography, Liquid; D002942:Circular Dichroism; D004247:DNA / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D019281:Dimerization; D004926:Escherichia coli / Q000235:genetics; D019077:Helix-Turn-Helix Motifs / Q000235:genetics; D008969:Molecular Sequence Data; D009154:Mutation; D010957:Plasmids / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D017421:Sequence Analysis; D017384:Sequence Deletion / Q000235:genetics; D013205:Staphylococcal Protein A; D013329:Structure-Activity Relationship; D015534:Trans-Activators / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D014357:Trypsin / Q000378:metabolism; D014764:Viral Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
D051778:Basic Helix-Loop-Helix Leucine Zipper Transcription Factors; C090591:C protein, Bacteriophage Mu; D004268:DNA-Binding Proteins; D011993:Recombinant Fusion Proteins; D013205:Staphylococcal Protein A; D015534:Trans-Activators; D014764:Viral Proteins; D004247:DNA; D014357:Trypsin