1998;283(1):71C81. filtration chromatography (Fig.2A). The different forms of both cleaved and uncleaved gp140s (monomer, dimer and trimer) were well resolved into distinct peaks on a Superose6 column. The uncleaved trimers migrated very slightly, but consistently, less rapidly than their cleaved counterparts Ombitasvir (ABT-267) but, overall, the two gp140 preparations had almost identical gel-filtration chromatography profiles. Open in a separate window Open in a separate window Fig. 2 Gel filtration analysis of cleaved and uncleaved gp140s. (A) The SOSIP.R6 G-MPER (cleaved) and SOSIP.IEGR G-MPER (uncleaved) gp140s were purified by lectin-affinity chromatography and then analyzed by size-exclusion chromatography. The separation profile of the two preparations over time (minutes) is shown. The solid arrow indicates the high-molecular aggregate peak, whereas the three dotted arrows show the trimer, dimer and monomer peaks. The precise timings (in minutes) of the three gp140 protomer peaks are listed. (B, C) The gel-filtration chromatography fractions (numbered above Ombitasvir (ABT-267) the lanes) were analyzed using BN-PAGE and western blotting. The migration positions of relevant molecular weight standards are marked on the left. A BN-PAGE analysis of fractions eluted from the gel filtration column again showed that the cleaved and uncleaved gp140s were very similar in their migration profiles (Fig.2B,C). However, higher molecular weight, disulphide-linked aggregates were present only in the cleaved gp140 preparation (Fig.2A, lane 1C4). These aggregates form a small shoulder just before the trimer maximum (denoted by a reddish arrow in Fig.2A). Since the purpose of these experiments was to compare cleaved and uncleaved trimers, we did not further analyze the dimeric, monomeric or other forms of gp140 that elute from your gel filtration column in and after portion 31; we have reported elsewhere on the presence of such Env forms in KNH1144 gp140 preparations (Dey et al., 2007). The peak related to the gp140 trimer portion was collected from your gel filtration column and concentrated to the desired volume. Subsequent recommendations to purified trimers refer to this gp140 portion, which we analyzed by ultracentrifugation and additional procedures as layed out below. Ultracentrifugation analysis Sedimentation equilibrium measurements were carried out to determine the oligomeric state of the purified, cleaved and uncleaved gp140s. The cleaved gp140 sedimented like a homogeous trimer; its apparent molecular mass is definitely ~460 kDa in the concentration range of 0.1 to 0.4 mg/ml (Fig.3A). The uncleaved gp140 is also trimeric, with an apparent molecular mass of ~435 kDa (Fig.3B). There was no systematic dependence of apparent molecular mass on protein concentration over a 4-fold range of protein concentration studied. Nonetheless, analysis of residual variations from your trimeric model reveals a systematic error, suggesting the uncleaved gp140 is definitely prone to aggregation. Open in a separate window Fig. 3 Sedimentation equilibrium analysis of cleaved and uncleaved gp140s. (A) Sedimentation equilibrium data of SOSIP.R6 G-MPER gp140 at 0.2 mg/ml in PBS (pH 7.0). Data are plotted as ln(absorbance) versus the square of the radius from your axis of rotation. The slope of Rabbit polyclonal to MBD3 the data is proportional to the molecular mass of the protein oligomer. SOSIP.R6 G-MPER gp140 is a discrete trimer under these conditions. (B) Sedimentation equilibrium data of SOSIP.IEGR G-MPER gp140 at 0.4 mg/ml in PBS (pH 7.0). The slope of the plotted data shows the SOSIP.IEGR G-MPER gp140 is a trimeric varieties. Stability of cleaved and uncleaved gp140 trimers We assessed Ombitasvir (ABT-267) the stabilities of the purified, cleaved and uncleaved gp140 trimers in the presence of different detergents, to gain an initial understanding of their properties. Both forms of trimer dissociated completely into monomeric gp140 subunits when incubated with the ionic detergent SDS for 1h at space heat (Fig.4A,B; lane 2). In the presence of non-ionic detergents (NP-40, Tween-20, Triton X-100), the uncleaved gp140 trimers were highly.