HIV-1 protease: mutagenesis of asparagine 88 indicates a domain required for dimer formation
Identifieur interne : 000403 ( France/Analysis ); précédent : 000402; suivant : 000404HIV-1 protease: mutagenesis of asparagine 88 indicates a domain required for dimer formation
Auteurs : Chantal Guenet [France] ; Ray A. Leppik [France] ; John T. Pelton [France] ; Karin Moelling [Allemagne] ; Walter Lovenberg [France] ; Bruce A. Harris [France]Source :
- European Journal of Pharmacology: Molecular Pharmacology [ 0922-4106 ] ; 1989.
English descriptors
- Teeft :
- Acad, Amino, Amino acids, Asparagine, Aspartate proteases, Aspartic acid, Bacterial extracts, Biochemical activity, Cleavage, Cleavage products, Clone, Codon, Coli, Complementary oligonucleotides, Complete nucleotide sequence, Core protein, Crystal structure, Enzymatic activity, Enzyme activity, Escherichia coli, Expression vector, Flow rate, Functional protease, Fusion protein, Immunodeficiency, Immunoreactive protein, Immunoreactivity, Iptg, Liquid chromatography, Monoclonal antibody, Mutagenesis, Mutant, Mutant enzyme, Mutant proteases, Natl, Oligonucleotides, Peptide, Precursor, Precursor proteins, Proc, Protease, Protease gene, Protease subunits, Proteolytic, Proteolytic activity, Proteolytic cleavage, Recombinant, Recombinant protease, Research institute, Retroviral proteases, Sequence analysis, Substitution, Synthetic gene, Synthetic oligonucleotides, Synthetic peptides.
Abstract
Abstract: Considerable interest exists in the HIV-1 protease for biochemical studies as a potential therapeutic target of acquired immunodeficiency syndrome. We have produced the retroviral enzyme in E. coli from a synthetic gene encoding the protease that was constructed by assembling six overlapping and complementary oligonucleotides into the vector pKK223-3. When expressed in E. coli, the recombinant protease was able to correctly process the HIV-1 core protein p24 from a β-galactosidase-gag fusion protein and to use a heptapeptide as a substrate for proteolytic cleavage. A single base pair mutation was identified in a recombinant that resulted in the substitution of lysine for asparagine at position 88 and a significant loss of enzyme activity. Through site-directed mutagenesis, the Asn88 was changed to five other residues representative of all classes of amino acids. The correlation between enzyme activity and amino acid substitution suggests that the protease domain surrounding position 88 affects the protein's potential for forming an active homodimeric protein and hence, indicates a biochemical interaction that could be inhibited by novel antiviral compounds.
Url:
DOI: 10.1016/0922-4106(89)90027-8
Affiliations:
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ISTEX:A1F4E0E99396B9E4B64652BCDD43A4CE4EED2330Le document en format XML
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Harris</name><affiliation wicri:level="3"><country xml:lang="fr">France</country><wicri:regionArea>Correspondence to: Bruce A. Harris, Merrell Dow Research Institute, 16 Rue d'Ankara, 67084 Strasbourg</wicri:regionArea><placeName><region type="region" nuts="2">Grand Est</region><region type="old region" nuts="2">Alsace (région administrative)</region><settlement type="city">Strasbourg</settlement></placeName></affiliation><affiliation wicri:level="3"><country xml:lang="fr">France</country><wicri:regionArea>Merrell Dow Research Institute, 16 Rue d'Ankara, 67084 Strasbourg</wicri:regionArea><placeName><region type="region" nuts="2">Grand Est</region><region type="old region" nuts="2">Alsace (région administrative)</region><settlement type="city">Strasbourg</settlement></placeName></affiliation></author></analytic><monogr></monogr><series><title level="j">European Journal of Pharmacology: Molecular Pharmacology</title><title level="j" type="abbrev">EJPMOL</title><idno type="ISSN">0922-4106</idno><imprint><publisher>ELSEVIER</publisher><date type="published" when="1989">1989</date><biblScope unit="volume">172</biblScope><biblScope unit="issue">6</biblScope><biblScope unit="page" from="443">443</biblScope><biblScope unit="page" to="451">451</biblScope></imprint><idno type="ISSN">0922-4106</idno></series></biblStruct></sourceDesc><seriesStmt><idno type="ISSN">0922-4106</idno></seriesStmt></fileDesc><profileDesc><textClass><keywords scheme="Teeft" xml:lang="en"><term>Acad</term><term>Amino</term><term>Amino acids</term><term>Asparagine</term><term>Aspartate proteases</term><term>Aspartic acid</term><term>Bacterial extracts</term><term>Biochemical activity</term><term>Cleavage</term><term>Cleavage products</term><term>Clone</term><term>Codon</term><term>Coli</term><term>Complementary oligonucleotides</term><term>Complete nucleotide sequence</term><term>Core protein</term><term>Crystal structure</term><term>Enzymatic activity</term><term>Enzyme activity</term><term>Escherichia coli</term><term>Expression vector</term><term>Flow rate</term><term>Functional protease</term><term>Fusion protein</term><term>Immunodeficiency</term><term>Immunoreactive protein</term><term>Immunoreactivity</term><term>Iptg</term><term>Liquid chromatography</term><term>Monoclonal antibody</term><term>Mutagenesis</term><term>Mutant</term><term>Mutant enzyme</term><term>Mutant proteases</term><term>Natl</term><term>Oligonucleotides</term><term>Peptide</term><term>Precursor</term><term>Precursor proteins</term><term>Proc</term><term>Protease</term><term>Protease gene</term><term>Protease subunits</term><term>Proteolytic</term><term>Proteolytic activity</term><term>Proteolytic cleavage</term><term>Recombinant</term><term>Recombinant protease</term><term>Research institute</term><term>Retroviral proteases</term><term>Sequence analysis</term><term>Substitution</term><term>Synthetic gene</term><term>Synthetic oligonucleotides</term><term>Synthetic peptides</term></keywords></textClass><langUsage><language ident="en">en</language></langUsage></profileDesc></teiHeader><front><div type="abstract" xml:lang="en">Abstract: Considerable interest exists in the HIV-1 protease for biochemical studies as a potential therapeutic target of acquired immunodeficiency syndrome. We have produced the retroviral enzyme in E. coli from a synthetic gene encoding the protease that was constructed by assembling six overlapping and complementary oligonucleotides into the vector pKK223-3. When expressed in E. coli, the recombinant protease was able to correctly process the HIV-1 core protein p24 from a β-galactosidase-gag fusion protein and to use a heptapeptide as a substrate for proteolytic cleavage. A single base pair mutation was identified in a recombinant that resulted in the substitution of lysine for asparagine at position 88 and a significant loss of enzyme activity. Through site-directed mutagenesis, the Asn88 was changed to five other residues representative of all classes of amino acids. The correlation between enzyme activity and amino acid substitution suggests that the protease domain surrounding position 88 affects the protein's potential for forming an active homodimeric protein and hence, indicates a biochemical interaction that could be inhibited by novel antiviral compounds.</div></front></TEI><affiliations><list><country><li>Allemagne</li><li>France</li></country><region><li>Alsace (région administrative)</li><li>Grand Est</li></region><settlement><li>Strasbourg</li></settlement></list><tree><country name="France"><region name="Grand Est"><name sortKey="Guenet, Chantal" sort="Guenet, Chantal" uniqKey="Guenet C" first="Chantal" last="Guenet">Chantal Guenet</name></region><name sortKey="Harris, Bruce A" sort="Harris, Bruce A" uniqKey="Harris B" first="Bruce A." last="Harris">Bruce A. Harris</name><name sortKey="Harris, Bruce A" sort="Harris, Bruce A" uniqKey="Harris B" first="Bruce A." last="Harris">Bruce A. Harris</name><name sortKey="Leppik, Ray A" sort="Leppik, Ray A" uniqKey="Leppik R" first="Ray A." last="Leppik">Ray A. Leppik</name><name sortKey="Lovenberg, Walter" sort="Lovenberg, Walter" uniqKey="Lovenberg W" first="Walter" last="Lovenberg">Walter Lovenberg</name><name sortKey="Pelton, John T" sort="Pelton, John T" uniqKey="Pelton J" first="John T." last="Pelton">John T. Pelton</name></country><country name="Allemagne"><noRegion><name sortKey="Moelling, Karin" sort="Moelling, Karin" uniqKey="Moelling K" first="Karin" last="Moelling">Karin Moelling</name></noRegion></country></tree></affiliations></record>Pour manipuler ce document sous Unix (Dilib)
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