A Promiscuous DNA Packaging Machine from Bacteriophage T4 (original) (raw)
Figure 2
Purification and characterization of phage heads.
(A) The 10am13am heads were isolated by differential centrifugation followed by CsCl gradient centrifugation (see Materials and Methods for details). The two closely spaced bands at the top of the gradient contained partial heads that had ejected most of their packaged DNA, save an ∼8-kb piece. The band at the bottom of the gradient contained full heads in which the packaged T4 genome was stabilized (see Figure 3 legend for additional details). (B) Purification of partial heads by DEAE-Sepharose column chromatography. The two closely spaced head bands at the top of the CsCl gradient were pooled, dialyzed against 10 mM Tris-HCl (pH 7.5), 50 mM NaCl, and 5 mM MgCl2, and purified by ion-exchange chromatography (AKTA Prime, GE Healthcare). The column was pre-equilibrated with 50 mM Tris-HCl (pH 7.5) and 5 mM MgCl2, and a linear gradient of 0–300 mM NaCl was applied to elute the bound heads. The peak fractions were pooled, concentrated by filtration, and stored at 4°C. (C) The partial and full heads are fully expanded. The purified proheads, partial heads, and full heads were mixed with SDS gel loading buffer and kept at room temperature (“−”) or boiling temperature (“+”) for 5 min. The samples were then separated by 10% SDS-PAGE, stained with Coomassie blue R, and destained. Note that the major capsid protein, gp23* (position marked with arrow), was not seen in the room temperature samples because the expanded heads could not be dissociated into gp23* subunits. (D) Partial and full heads reassemble with the exogenous gp17. About 5×1011 proheads, partial heads, or full heads were incubated with purified gp17-K577 (0.3 µM; 50:1 ratio of gp17 molecules to gp20 subunits) in a buffer containing 50 mM Tris-HCl (pH 7.5), 100 mM NaCl, and 5 mM MgCl2, at room temperature for 30 min. The head-gp17 complexes were sedimented by centrifugation at 18,000 rpm for 45 min, and the pellet was washed several times to remove any unbound gp17. The proteins were transferred to PVDF membrane, and Western blotting was performed using polyclonal gp17 antibodies. The results were confirmed by doing the same experiment with full-length gp17 and a GFP-gp17 fusion protein. Only the gp17-K577 (C-terminal 33 amino acids of gp17 were deleted) data are shown because gp17-K577 is protease resistant and migrates as a single band as opposed to three bands with the full-length gp17 and GFP-gp17, and also because there is no background overlapping band at the same position. The gp17 band in the full head lane (lane 4) is faint because some of these heads released the packaged DNA during the procedure, which resulted in poor recovery of the heads during the centrifugation and washing steps.