HIV-1 Aspartic Proteinase: High-Level Production and Automated Fluorometric Screening Assay of Inhibitors

Summary 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 mater­ ial after sonication of the bacteria was composed of aggregated proteinase. Subsequent renaturation and purification yielded largequantities of a homogeneous enzyme able to cleave various heptapeptidic substrates in vitro with a Km 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 intramolecularlyquenched fluorogenic substrate Suc­ TLNFPIS-4MCA based on the heptapeptide TLNFPIS, which encompasses the proteinase/reverse trans­ criptasejunction, 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 microplatefluorescence scanner. The known aspartic proteinase inhibitor pepstatin A consistentlygaveI( = 2 x 10- 6 M. Other synthetic and natural compounds