A defined system for in vitro packaging of DNA-gp3 of the Bacillus subtilis bacteriophage Φ29

The bacteriophage Φ29 DNA-gene product 3 complex (DNA-gp3) has been efficiently packaged into proheads in a completely defined in vitro system. The Φ29 DNA packaging protein gp16, the product of gene 16, was overproduced in Escherichia coli and purified to near homogeneity. The purified gp16 packaged 23% of the DNA-gp3 added to purified proheads in the defined mixture, while gp16 in an extract of phage-infected cells packaged 26% of the DNA-gp3. No host proteins were required in the defined system. ATP-dependent packaging of DNA-gp3 in the defined system was optimal with ~ 100 copies of gp16 per DNA-gp3, an amount similar to the production of gp16 per DNA-gp3 in Φ29-infected cells.