In vitro assembly of the Bacillus subtilis bacteriophage phi 29

In vitro assembly of the Bacillus subtilis bacteriophage phi 29 that approaches the efficiency of assembly in vivo has been demonstrated. Proheads, DNA, and gene 16 product (gp16) were essential for DNA encapsidation, and the average yield in extracts was 180 phage per prohead donor cell. The in vitro maturation was very similar to in vivo assembly in terms of yield, intermediates, and abortive structures. More that 30% of the proheads in the extract were converted to phage, and about 20% of DNA--protein extracted from phage could be repackaged. In vitro assembly was blocked by the addition of DNase I, EDTA, pyrophosphatase, or the ATP analogues adenosine 5'-[alpha, beta-methylene]triphosphate and adenosine 5'-[beta, gamma-methylene]triphosphate. Less than 1% of the proheads isolated in sucrose gradients can accept DNA--protein in packaging in vitro.