title: Assay of HIV-1 protease activity by use of crude preparations of enzyme and biotinylated substrate.
abstract: 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 expression 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. coli, fused with a C-terminal peptide that contains a protease cleavage site flanked by p17 and p24 segments. Through its biotin in the N-terminal region, Pin17/24 bound to ELISA plates coated with avidin, whereas through its C-terminal region, the same molecule of Pin17/24 could be recognized by an anti-p24 monoclonal antibody. When the protease was added to Pin17/24, the p24 fragment was released from the biotinylated fusion protein and could no longer be retained on the avidin plates, and as a result, binding of the anti-p24 monoclonal antibody decreased. The binding was specific and the reaction was inhibited by a known HIV protease inhibitor. Due to the specific interactions between avidin and biotin, monoclonal antibody and antigen, and the HIV protease and the gag substrate, crude preparations of these reagents can be used readily in the assay. The simplicity and feasibility of this method should be useful for simultaneous monitoring of many enzyme reactions, particularly for screening possible HIV protease inhibitors.
authors.affiliation: Graduate Institute of Microbiology and Immunology, National Yang-Ming University, Taipei, Taiwan, ROC.
authors.forename: S L
authors.initials: SL
authors.lastname: Yu
authors.forename: N
authors.initials: N
authors.lastname: Wang
authors.forename: C Y
authors.initials: CY
authors.lastname: Liou
authors.forename: W J
authors.initials: WJ
authors.lastname: Syu
mesh_terms: D000818:Animals; D001710:Biotin; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D004926:Escherichia coli; D005240:Feasibility Studies; D015683:Gene Products, gag / Q000378:metabolism*; D016333:HIV Protease / Q000032:analysis*; D015497:HIV-1 / Q000201:enzymology*; D006801:Humans; D051379:Mice; D008807:Mice, Inbred BALB C; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013379:Substrate Specificity
chemical_list: D015683:Gene Products, gag; D011993:Recombinant Fusion Proteins; D001710:Biotin; D016333:HIV Protease