OVERPRODUCTION AND PURIFICATION OF C-PROTEIN, THE LATE GENE-TRANSCRIPTION ACTIVATOR FROM PHAGE-MU

We report here the high-level overproduction and single-step purificat ion for the C protein of bacteriophage Mu. Attempts to secrete the pro tein using the pelB signal sequence, in a T7 expression system, failed to yield the processed product. Moreover, the overexpressed fusion pr otein was inactive in DNA binding assays. In order to obtain the nativ e protein, the sequences coding for the signal peptide were removed. T he clones thus obtained upon induction overproduced the C protein, a s ignificant amount of which was present in the S20 pellet fraction. The protein was recovered from this pellet by high salt extraction and pu rified 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. (C) 1994 Academ ic Press, Inc.