| Autor: |
Hoffmann, Dieter, Assfalg-Machleidt, Irmgard, Nitschko, Hans, Von der Helm, Klaus, Koszinowski, Ulrich, Machleidt, Werner |
| Předmět: |
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| Zdroj: |
Biological Chemistry; Jul2003, Vol. 384 Issue 7, p1109-1117, 9p, 5 Charts, 2 Graphs |
| Abstrakt: |
A phenotypic resistance test based on recombinant expression of the active HIV protease in E. coli from patient blood samples was developed. The protease is purified in a rapid one-step procedure as active enzyme and tested for inhibition by five selected synthetic inhibitors (amprenavir, indinavir, nelfinavir, ritonavir, and saquinavir) used presently for chemo-therapy of HIV-infected patients. The HPLC system used in a previous approach was replaced by a continuous fluorogenic assay suitable for high-throughput screening on microtiter plates. This reduces significantly the total assay time and allows the determination of inhibition constants (K[subi]). The Michaelis constant (K[subm]) and the inhibition constant (K[subi]) of recombinant wild-type protease agree well with published data for cloned HIV protease. The enzymatic test was evaluated with recombinant HIV protease derived from eight HIV-positive patients scored from 'sensitive' to 'highly resistant' according to mutations detected by genotypic analysis. The measured K(subi) values correlate well with the genotypic resistance scores, but allow a higher degree of differentiation. The non-infectious assay enables a more rapid yet sensitive detection of HIV protease resistance than other phenotypic assays. [ABSTRACT FROM AUTHOR] |
| Databáze: |
Complementary Index |
| Externí odkaz: |
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