In vitro Assay of Packaging Protein gp3 of <i>Salmonella</i> Phage P22

Abstract

The packaging proteins, gp2 and gp3, of Salmonella phage P22 were separated by DEAE-sephacel column chromatography. The activity of both proteins was measured by complementation in an in vitro phage packaging assay. For the first time gp3 activity was demonstrated in vitro. The majority of the gp3 activity could be separated from gp2. While gp2 was stable during chromatography and storage, gp3 was very unstable. However, it was stable when stored as a crude extract. The ratio and interaction of these two proteins critical for phage maturation are still uncertain.

Cloning and Expression of Packaging Gene 3 of <i>Salmonella</i> Phage P22

“…Difficulties to isolate the packaging pro tein gp3 of Salmonella phage P22 and to perform in vitro studies [3] stimulated the approach to clone gene 3 in an expression vector. Such clones, in combination with gene 3 defective phages, allowed the differ ential assay of gp3 activity and of the target site of gp3 (the pacsite).…”

“…Gene product (gp) 3 is responsible for recognition of the pac site at which sequential packaging of headfulsized fragments from a concatemeric precur sor DNA starts. In vitro studies of gp3 are extremely difficult because in phage-infec ted cells this protein is expressed only in minimal quantities [I, 2], Its biological activ ity is also lost very quickly during purifica tion [3]. To overcome these obstacles, gene 3 (of P22 wild-type phage and of two mutants with altered packaging properties) was cloned into an expression vector.…”

Viral Small Terminase: A Divergent Structural Framework for a Conserved Biological Function

“…However, in most of the reaction steps described above, TerS is not part of the packaging motor, or at least, in vitro, does not appear to play a direct role in genome packaging. For instance, bulk genome packaging systems developed for phages T4 [ 34 ], T3 [ 35 ], λ [ 36 ], and P22 [ 15 , 37 , 38 ] did not require TerS in vitro. Similarly, TerS inhibited packaging in a defined in vitro packaging system carried out in the presence of purified T4 [ 39 , 40 , 41 ] or SPP1 [ 42 ] components.…”

Function and horizontal transfer of the small terminase subunit of the tailed bacteriophage Sf6 DNA packaging nanomotor

“…In apparent contradiction to this view, removal of twenty C-terminal amino acids, which make up most of the C-terminal tubular β-barrel domain of the P22 TerS, does not impair its oligomerization but does block its DNA binding capability (Nemecek et al ., 2008; Roy et al ., 2012). T4 TerS is dispensable in a T4 in vitro DNA packaging system (Al-Zahrani et al ., 2009; Zhang et al ., 2011), while in P22 and SPP1 TerS protein is required but specific recognition of the packaging target site is not required in vitro (Poteete and Botstein, 1979; Schmieger, 1984; Schmieger and Koch, 1987; Oliveira, Alonso, and Tavares, 2005). Thus much remains to be understood regarding small terminase subunit function and its role in DNA packaging motor initiation.…”