HIV-1 protease variants from 100-fold drug resistant clinical isolates: expression, purification, and crystallization

Vickrey, John F.; Logsdon, Bradley C.; Proteasa, Gheorghe; Palmer, Sarah; Winters, Mark A.; Merigan, Thomas C.; Kovari, Ladislau C.

doi:10.1016/s1046-5928(02)00650-2

Cited by 20 publications

(24 citation statements)

References 37 publications

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“…Freshly transformed Escherichia coli strain BL21(DE3) (with the T7 expression vector containing the MDR 769 HIV-1 protease clinical isolate) was grown in liquid yeast tryptone ampicillin medium at 37°C in a shaking incubator, and the mid-log-phase bacterial culture was used to inoculate five 1-liter YT ampicillin medium flasks. This bacterial culture continued to grow for 18 to 20 h after inoculation (52).…”

Section: Methodsmentioning

confidence: 99%

“…The MDR isolate 769 HIV-1 protease was purified and refolded from inclusion bodies, following previously published protocol (52). Briefly, after cell lysis the soluble protein fraction was removed from the inclusion bodies and discarded.…”

Section: Methodsmentioning

confidence: 99%

“…The crystals were bipyramidal in morphology and formed from 2-l drops at 22°C (52). After at least 14 days from the completion of the protease crystal formation, protease-inhibitor complexes were obtained by adding a small amount of solid inhibitor directly to the drop containing the protease crystals.…”

Section: Methodsmentioning

confidence: 99%

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Crystal Structures of a Multidrug-Resistant Human Immunodeficiency Virus Type 1 Protease Reveal an Expanded Active-Site Cavity

Logsdon¹,

Vickrey²,

Martin³

et al. 2004

J Virol

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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Crystal Structures of a Multidrug-Resistant Human Immunodeficiency Virus Type 1 Protease Reveal an Expanded Active-Site Cavity

Logsdon¹,

Vickrey²,

Martin³

et al. 2004

J Virol

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“…Second, the HIV protease gene in p1ϩIQ was PCR amplified, subcloned into pET28aϩ using NdeI and Hind III, and sequenced to confirm its wild-type identity. The subcloning fused DNA encoding a hexahistidine tag to its 5Ј-end; this tag does not affect enzyme activity (31). The inactivating D25N mutation was introduced into His 6 -HIV PR-pET28 by whole circle PCR using primers 5Ј-GAAGCTCTATTA-AATACAGGAGCAGATG-3Ј (HIVPR-D25N-62) and 5Ј-CTTTAGT-TGCCCCCCTATCTTTATTGTG-3Ј (HIVPR-62out).…”

Section: Methodsmentioning

confidence: 99%

“…The total protein concentration was quantified using the Bradford protein assay (BioRad). HIV protease was purified and refolded according to an established protocol (31) and protein concentration also quantified using the Bradford protein assay. We used a commercially available fluorogenic substrate to show that our HIV protease was as active as those described in the literature (33).…”

Section: Methodsmentioning

confidence: 99%

Rational Design of p53, an Intrinsically Unstructured Protein, for the Fabrication of Novel Molecular Sensors

Geddie

O'Loughlin

Woods

et al. 2005

Journal of Biological Chemistry

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The dominant paradigm of protein engineering is structurebased site-directed mutagenesis. This rational approach is generally more effective for the engineering of local properties, such as substrate specificity, than global ones such as allostery. Previous workers have modified normally unregulated reporter enzymes, including ␤-galactosidase, alkaline phosphatase, and ␤-lactamase, so that the engineered versions are activated (up to 4-fold) by monoclonal antibodies. A reporter that could easily be "reprogrammed" for the facile detection of novel effectors (binding or modifying activities) would be useful in high throughput screens for directed evolution or drug discovery. Here we describe a straightforward and general solution to this potentially difficult design problem. The transcription factor p53 is normally regulated by a variety of post-translational modifications. The insertion of peptides into intrinsically unstructured domains of p53 generated variants that were activated up to 100-fold by novel effectors (proteases or antibodies). An engineered p53 was incorporated into an existing high throughput screen for the detection of human immunodeficiency virus protease, an arbitrarily chosen novel effector. These results suggest that the molecular recognition properties of intrinsically unstructured proteins are relatively easy to engineer and that the absence of crystal structures should not deter the rational engineering of this class of proteins.Cells generally employ sensor proteins (also called "biosensors" or "switches") to detect chemical stimuli and activate downstream components of signal transduction systems. We sought to fabricate artificial molecular sensors by engineering proteins that are specifically activated when bound or modified by novel effectors. Such sensors have practical utility in high throughput screens for drug discovery or directed protein evolution. They have also proved to be useful as research reagents. For example, two-hybrid systems (1, 2) and protein fragment complementation assays (3) couple the interactions of fusion proteins within transgenic cells to the production of signals. Cleverly designed sensors based upon fluorescent resonance energy transfer between green fluorescent protein analogues have also enabled the observation of intracellular protein modification events (4), including protein phosphorylation (5) and proteolysis (6). We expect that the utility of engineered protein sensors will continue to increase as they are deployed as diagnostic reagents (7) and pathogen-activated biotherapeutics (8, 9).Rational protein design is generally synonymous with structure based site-directed mutagenesis (10). Reporter proteins are usually selected as starting points for sensor design because their structurehave been solved and because their activities are amenable to high throughput screening. Previous workers have inserted peptide epitopes into ␤-galactosidase (11), alkaline phosphatase (12), or ␤-lactamase (13). This approach has generally produced catalytically compromis...

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Nine Crystal Structures Determine the Substrate Envelope of the MDR HIV-1 Protease

et al. 2011

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Under drug selection pressure, emerging mutations render HIV-1 protease drug resistant, leading to the therapy failure in anti-HIV treatment. It is known that nine substrate cleavage site peptides bind to wild type (WT) HIV-1 protease in a conserved pattern. However, how the multidrug-resistant (MDR) HIV-1 protease binds to the substrate cleavage site peptides is yet to be determined. MDR769 HIV-1 protease (resistant mutations at residues 10, 36, 46, 54, 62, 63, 71, 82, 84, and 90) was selected for present study to understand the binding to its natural substrates. MDR769 HIV-1 protease was co-crystallized with nine substrate cleavage site hepta-peptides. Crystallographic studies show that MDR769 HIV-1 protease has an expanded substrate envelope with wide open flaps. Furthermore, ligand binding energy calculations indicate weaker binding in MDR769 HIV-1 protease-substrate complexes. These results help in designing the next generation of HIV-1 protease inhibitors by targeting the MDR HIV-1 protease.

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HIV-1 protease variants from 100-fold drug resistant clinical isolates: expression, purification, and crystallization

Cited by 20 publications

References 37 publications

Crystal Structures of a Multidrug-Resistant Human Immunodeficiency Virus Type 1 Protease Reveal an Expanded Active-Site Cavity

Crystal Structures of a Multidrug-Resistant Human Immunodeficiency Virus Type 1 Protease Reveal an Expanded Active-Site Cavity

Rational Design of p53, an Intrinsically Unstructured Protein, for the Fabrication of Novel Molecular Sensors

Nine Crystal Structures Determine the Substrate Envelope of the MDR HIV-1 Protease

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