Characterization, overproduction and purification of the product of gene 1 of Bacillus subtilis phage ø29

Abstract

Unit-length φ29 DNA was not synthesized after restrictive infection of Bacillus subtilis with the φ29 mutant sus 1(629) indicating that the phage φ29 protein p1 is needed for the viral DNA replication. Sequencing of the ORF-6 of mutant sus1 (629) showed that a C in the wild-type (wt) phage had been changed to a T at nt position 19 of the ORF-6, giving rise to a TAA ochre codon, indicating that this ORF corresponds to gene 1. ORF-6 was cloned in plasmid pPLc28 under the control of the pL promoter of phage λ and, after induction, a protein of about 10 kDa was overproduced, which was absent in the corresponding cells harbouring a recombinant plasmid with the sus1 (629) mutation, indicating that the 10-kDa protein is the product of gene 1. In addition, a protein of lower Mr was synthesized after induction of the cells harbouring recombinant plasmids with the wt or the sus1 (629) DNA. Both proteins were purified and characterized by N-terminal sequence determination and amino acid analysis. The low-Mr protein, named Δ1, has a size of 6 kDa and corresponds to an internal in-phase initiation event in ORF-6.