Figure 5

Analysis of host vesicles. (a) Host vesicles were prepared as in Figure 2, but using a lysate of E. coli BL21 infected with T7 amber mutant in genes 5 (DNA polymerase) and 19 (DNA packaging ATPase). Proteins in a fraction with density of 1.265 g/ml were then stained with 80 μg/ml Alexa 488 at 21°C for 2 hr. in 0.09 M Tris-Acetate, pH 8.4, 0.001 M MgCl₂ (electrophoresis buffer). Alexa-stained particles were subjected to 2d-AGE [19] in electrophoresis buffer at room temperature (25 ± 3°C) through one of four 0.3% first dimension gels that were embedded in a 1.8% second dimension gel [19]. First dimension: 2.0 V/cm, 3.8 hr; second dimension: 1.8 V/cm, 8.5 hr. Alexa 488 fluorescence was observed under ultraviolet illumination, photographed and used for slicing the agarose gel (slices A-E). Capsid I (radius = 26.1 nm) was fractionated in another first dimension gel embedded in the same second dimension gel. The band of capsid I (CI), positioned accurately, is overlaid in a box. Arrowheads indicate origin; arrows outside of the gel indicate directions of first (I) and second (II) dimension electrophoresis. The dashed line is drawn from the origin through the CI band; particles on this line have R_E of 26.1 nm. (b) The slices described above were subjected to SDSPAGE in a 10% gel and stained with SYPRO Ruby protein gel stain (Bio-Rad). Proteins from selected bands were digested with trypsin and analyzed by capillary HPLC-tandem mass spectrometry, as previously described [25]. Protein identity is indicated at the right. The proteins marked with an asterisk were present in the background, as well as the stained agarose gel regions, for reasons not known. The presence in the background of a chaperonin is, however, not surprising because of the known non-specific and reversible protein-chaperonin binding.