Preparation of recombinant HIV-1 gag protein and assembly of virus-like particles in vitro

Abstract

The mechanism of assembly of retroviruses is not fully understood. Purification of retroviral Gag protein and studying its solution state and assembly properties might provide insights into retroviral assembly mechanisms. Here we describe a rapid method for the purification of Gag and its subsequent assembly into virus-like particles in a defined system in vitro. The purification scheme does not use affinity tags, but purifies the native protein by virtue of its high affinity for phosphocellulose, a property presumably related to the affinity of Gag proteins for nucleic acids.

Fig. 14.1.

Electron micrograph of VLPs assembled from Δ16–99 p6 Gag by dilution method, as described. Samples were negatively stained with 2% uranyl acetate. Note the heterogeneity in morphology, including some incompletely assembled VLPs.

Fig. 14.2.

SDS-PAGE analysis of different stages of the purification procedure. The arrow on the left shows the position of Gag on the gel. Lanes 1: supernatant from step 3.4.5 after sonication and centrifugation (8 μL); 2: step 3.4.9 before centrifugation (5 μL); 3: step 3.4.10 after centrifugation (5 μL); 4: supernatant from step 3.4.15 – unbound material from PC (25 μL); 5: step 3.4.16 −0. 1 M NaCl wash of PC (25 μL); 6: step 3.4.17 −0. 2 M NaCl wash of PC (25 μL); 7: step 3.4.18 0. 5 M NaCl wash of PC (20 μL); 8 (4 μL) and 9 (20 μL) of pooled fractions from step 3.4.19 to 22 −1 M NaCl washes; 10: molecular weight markers. M.W. indicated next to bands in kDa. Note that some Gag failed to bind the PC at step 3.4.15, but this loss is offset by the fact that a very large fraction of the contaminating proteins has been eliminated here (lane 4).