Sephadex g 75 superfine

The expression, isolation, and purification of NL4–3, NL43(L89V), NL4–3(L90M), NL4–3(DM), and KY variants used for all assays were carried out as previously described^{46 (link)–47 (link)}. Briefly, the gene encoding the desired HIV protease was subcloned into the heat-inducible pXC35 expression vector (ATCC) and transformed into E. coli TAP-106 cells. Cells grown in 6L of Terrific Broth (TB) were lysed with a cell disruptor twice and the protein was purified from inclusion bodies^{50 (link)}. The inclusion body centrifugation pellet was dissolved in 50% acetic acid followed by another round of centrifugation at 19K for 30 minutes to remove impurities. Size exclusion chromatography was carried out on a 2.1-L Sephadex G-75 superfine (Sigma Chemical) column equilibrated with 50% acetic acid to separate high molecular weight proteins from the desired protease. The cleanest fractions of HIV protease were refolded into a 10fold dilution of refolding buffer [0.05 M sodium acetate at pH 5.5, 5% ethylene glycol, 10% glycerol, and 5 mM dithiothreitol (DTT)]. Folded protein was concentrated down to 0.5–3mg/mL and stored. The stored protease was used in kinetics and binding assays.