ASSAY OF HIV-1 PROTEASE ACTIVITY BY USE OF CRUDE PREPARATIONS OF ENZYME AND BIOTINYLATED SUBSTRATE

An enzyme immunoassay was developed for monitoring protease reactions of human immunodeficiency virus (HIV). The protease and its substrate, the gag precursor, were generated separately in Escherichia coli. The HIV-1 protease was generated with a glutathione-S-transferase express ion system and the gag substrate, named Pin17/24, was prepared with a PinPoint expression system. Pin17/24 consists of an N-terminal peptide , which is biotinylated in E. coil, fused with a C-terminal peptide th at contains a protease cleavage site flanked by p17 and p24 segments. Through its biotin in the N-terminal region, Pin17/24 bound to ELISA p lates coated with avidin, whereas through its C-terminal region, the s ame molecule of Pin17/24 could be recognized by an anti-p24 monoclonal antibody. When the protease was added to Pin17/24, the p24 fragment w as released from the biotinylated fusion protein and could no longer b e retained on the avidin plates, and as a result, binding of the anti- p24 monoclonal antibody decreased. The binding was specific and the re action was inhibited by a known HIV protease inhibitor. Due to the spe cific interactions between avidin and biotin, monoclonal antibody and antigen, and the HIV protease and the gag substrate, crude preparation s of these reagents can be used readily in the assay. The simplicity a nd feasibility of this method should be useful for simultaneous monito ring of many enzyme reactions, particularly for screening possible HIV protease inhibitors.