Purification and characterization of the human immunodeficiency virus type 1 integrase expressed in Escherichia coli

The integrase protein mediates insertion of viral DNA into cellular DNA that is essential for viral replication and virion production. The integrase: protein of the human immunodeficiency virus type-1 (HIV-1) was overexpressed from E. coli, purified by using nickel-chelated column in a one-step manner, and characterized by monitoring the endonucleolytic activity to investigate whether it can be directly used in inhibitor-screening. Active integrase was eluted from the column at 100 mM imidazole. The endonucleolytic activity of the integrase protein was studied in various conditions. The endonucleolytic activity was not inhibited at up to 35% glycerol while it was significantly inhibited at dimethyl-sulfoxide of more than 10%. CHAPS that was used to dissolve the integrase protein from bacterial lysate pellets did not inhibit the activity under 10 mM. Therefore, it is suggested that the integrase protein eluted with 100 mM imidazole buffer containing 25 mM 3-[(3-cholamidopropyl)-dimethylammonio]-1-propane-sulfonate (CHAPS) could be directly used in inhibitor-screening by mixing one volume of the enzyme solution with 9 volumes of the reaction buffer containing oligonucleotide substrate. In addition, endonucleolytic activity was not inhibited in the presence of NaCl of less than 175 mM, KCI of less than 100 mM, or CaCl2 of less than 50 mM. Mn++ ion, known as a cofactor, did not reduce the activity at up to 22 mM tested while Mg++ ion decreased the activity remarkedly above 10 mM.