HIV-1 aspartic proteinase: high-level production and automated fluorometric screening assay of inhibitors

The 99-amino-acid HIV-1 aspartic proteinase was expressed to high levels in Escherichia coli using a T7 expression system. About 50% of the insoluble material after sonication of the bacteria was composed of aggregated proteinase. Subsequent renaturation and purification yielded large quantities of a homogeneous enzyme able to cleave various heptapeptidic substrates in vitro with a K-m around 2.5 mM. A fluoro-metric assay has been devised to allow automated screening of HIV proteinase inhibitors based on an analogous renin assay. We used the synthetic intramolecularly quenched fluorogenic substrate Suc-TLNFPIS-4MCA based on the heptapeptide TLNFPIS, which encompasses the proteinase/reverse transcriptase junction, coupled to the fluorophore 7-amino-4-methylcoumarin and blocked at the amino-terminus by a succinyl group. The enzyme cleaves the substrate between phenylalanine and proline, and conditions were optimized for liberation of 7AMC from the generated PIS-4MCA with aminopeptidase M as secondary enzyme. 7AMC was monitored with a microplate fluorescence scanner. The known aspartic proteinase inhibitor pepstatin A consistently gave K-i = 2 x 10(-6) M. Other synthetic and natural compounds are currently being tested.