HIV-1 Aspartic Proteinase: High-Level Production and Automated Fluorometric Screening Assay of Inhibitors
| Autor: | B. Cambou, Yves Lelièvre, C. Giuliacci, Terence Cartwright, G. Jung, A. Dugué, Fabienne Parker, J. Boiziau, J.-F. Mayaux, Ph.-H. Hirel, D. Outerovitch, C. Peltiers, R. Boulay |
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| Rok vydání: | 1990 |
| Předmět: |
0301 basic medicine
chemistry.chemical_classification biology 030106 microbiology Fluorescence spectrometry Substrate (chemistry) Phenylalanine General Medicine medicine.disease_cause 01 natural sciences 0104 chemical sciences 010404 medicinal & biomolecular chemistry 03 medical and health sciences chemistry.chemical_compound Enzyme chemistry Biochemistry Biosynthesis Enzyme inhibitor biology.protein medicine Escherichia coli Pepstatin |
| Zdroj: | Antiviral Chemistry and Chemotherapy. 1:9-15 |
| ISSN: | 2040-2066 |
| DOI: | 10.1177/095632029000100103 |
| Popis: | 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 Km around 2.5 mM. A fluorometric 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 Ki = 2 × 10−6M. Other synthetic and natural compounds are currently being tested. |
| Databáze: | OpenAIRE |
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