Recombinant Purification of HIV Nucleocapsid Proteins

NCp7 and NCp8 proteins were purified as recombinant glutathione S-transferase (GST) fusion proteins from E. coli BL21—Codon Plus (DE3)—RIL cells (Stratagene), based on engineered expression vectors pGEX-4 T-3-NCp7 and pGEX-4 T-3-NCp8 [58 (link)]. The HIV-1_NL4–3 and HIV-2_ROD nucleocapsid protein coding sequencescoli BL21—Codon Plus (DE3)—RIL cells (Stratagene), based on engineered expression vectors pGEX-4 T-3-NCp7 and pGEX-4 T-3-NCp8 [58 (link)]. The nucleocapsid protein coding sequences of HIV-1_NL4–3 and HIV-2_ROD were taken from http://ncbi.nlm.nih.gov. GST-tagged proteins were purified by affinity chromatography on Glutathione-Sepharose (Amersham Pharmacia Biotech.) and the GST tag was cleaved off with thrombin enzyme. The proteins were further purified on a Superdex 200 FPLC column, and molecular masses were determined using MALDI TOF (Autoflex, Bruker Daltonics). The purified proteins were lyophilized and stored at −80°C. Synthetic (8–48) NCp8 peptide was obtained from ThermoFischer Scientific. Proteins were dissolved in freshly prepared, oxygen-free buffer containing 30 mM HEPES pH 6.5, 30 mM NaCl and 0.1 mM ZnCl₂. Activity of each protein preparation was compared with the activity of the synthetic full-length (48aa) NCp8 (Additional file 2: Figure S2).