Large-Scale Functional Purification of Recombinant HIV-1 Capsid
Figure 2
Solution oligomeric state of functionally purified WT CA and assembled CA 4Mu hexamer.
(A) Full-length wild-type capsid protein (WT CA) and (B) hexameric capsid construct obtained by disulfide cross-linking of CA 4Mu monomer ([A14C,E45C,W184A,M185A]CA). Sedimentation velocity experiments were performed in 50 mM sodium phosphate buffer (pH 7.5) at 25°C and 45,000 rpm. Absorbance scans at 280 nm were collected for CA FL (A) at 0.94 µM, 1.88 µM, 3.75 µM, and 7.5 µM, and for CA 4Mu Hexamer (B) at 4.13 µM, 8.25 µM (±1 mM TCEP) and 16.5 µM (expressed as monomers of CA 4Mu). Sedimentation coefficient distribution – c(s) - was calculated using Sedfit program. WT CA contains monomers in equilibrium with dimers, whereas CA 4Mu hexamer preparation contains 94% of 4Mu monomers in hexameric form and 6% as a mixture of monomers, dimers and trimers. Treatment with 1 mM TCEP converts hexamers back to 4Mu CA monomers.