Overproduction and purification of C protein, the late gene transcription activator from phage Mu.
We report here the high-level overproduction and single-step purification for the C protein of bacteriophage Mu. Attempts to secrete the protein using the pelB signal sequence, in a T7 expression system, failed to yield the processed product. Moreover, the overexpressed fusion protein was inactive in DNA binding assays. In order to obtain the native protein, the sequences coding for the signal peptide were removed. The clones thus obtained upon induction overproduced the C protein, a significant amount of which was present in the S20 pellet fraction. The protein was recovered from this pellet by high salt extraction and purified by specific immunoaffinity chromatography. The purified protein was active in DNA binding assay. The final yield of the protein was 9 mg of approximately 95% purity from 1 g wet wt cells.
Ramesh
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Centre for Genetic Engineering, Indian Institute of Science, Bangalore.
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Nagaraja
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D010583:Bacteriophage mu; D001483:Base Sequence; D051778:Basic Helix-Loop-Helix Leucine Zipper Transcription Factors; D002846:Chromatography, Affinity; D004268:DNA-Binding Proteins / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification*; D015967:Gene Expression Regulation, Viral; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification; D015534:Trans-Activators / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification*; D014158:Transcription, Genetic; D014764:Viral Proteins
D051778:Basic Helix-Loop-Helix Leucine Zipper Transcription Factors; C090591:C protein, Bacteriophage Mu; D004268:DNA-Binding Proteins; D011994:Recombinant Proteins; D015534:Trans-Activators; D014764:Viral Proteins