PURIFICATION AND CHARACTERIZATION OF THE HUMAN-IMMUNODEFICIENCY-VIRUSTYPE-1 INTEGRASE EXPRESSED IN ESCHERICHIA-COLI

The integrase protein mediates insertion of viral DNA into cellular DN A that is essential for viral replication and virion production. The i ntegrase: protein of the human immunodeficiency virus type-1 (HIV-1) w as overexpressed from E. coli, purified by using nickel-chelated colum n in a one-step manner, and characterized by monitoring the endonucleo lytic activity to investigate whether it can be directly used in inhib itor-screening. Active integrase was eluted from the column at 100 mM imidazole. The endonucleolytic activity of the integrase protein was s tudied in various conditions. The endonucleolytic activity was not inh ibited at up to 35% glycerol while it was significantly inhibited at d imethyl-sulfoxide of more than 10%. CHAPS that was used to dissolve th e integrase protein from bacterial lysate pellets did not inhibit the activity under 10 mM. Therefore, it is suggested that the integrase pr otein eluted with 100 mM imidazole buffer containing 25 mM lamidopropy l)-dimethylammonio]-1-propane-sulfonate (CHAPS) could be directly used in inhibitor-screening by mixing one volume of the enzyme solution wi th 9 volumes of the reaction buffer containing oligonucleotide substra te. In addition, endonucleolytic activity was not inhibited in the pre sence of NaCl of less than 175 mM, KCI of less than 100 mM, or CaCl2 o f less than 50 mM. Mn++ ion, known as a cofactor, did not reduce the a ctivity at up to 22 mM tested while Mg++ ion decreased the activity re markedly above 10 mM.