Sup35p yeast prion-like protein as an adapter for production of the Gag-p55 antigen of HIV-1 and the L-chain of botulinum neurotoxin in Saccharomyces cerevisiae
Identifieur interne : 003509 ( Main/Curation ); précédent : 003508; suivant : 003510Sup35p yeast prion-like protein as an adapter for production of the Gag-p55 antigen of HIV-1 and the L-chain of botulinum neurotoxin in Saccharomyces cerevisiae
Auteurs : Pavel A. Ivanov [Russie] ; Eugene I. Lewitin [Russie] ; Boris I. Shevelev [Russie] ; Gleb V. Fominov [Russie] ; Jana A Wojciechowska [Russie] ; Ali H. Asadi Mobarhan [Russie] ; Yuri V. Vertiev [Russie] ; Nick K. Yankovsky [Russie] ; A. B Shevelev [Russie]Source :
- Research in Microbiology [ 0923-2508 ] ; 2001.
English descriptors
- KwdEn :
- Teeft :
- Alkaline phosphatase, Amino acid residues, Bamhi, Bont, Botl, Botulinum neurotoxin, Bst1, Cerevisiae, Cerevisiae strain, Cerevisiae transformation, Chimeric, Chimeric gene, Chimeric protein, Cold spring harbor laboratory press, Coli, Derivative, Empty vector, Encoding, Escherichia coli, Fusion protein, Galactose induction, Gene, Gene encoding, Gene expression, Growth rate, Gure, Heterologous, Human virus type, Immunopositive product, Inclusion bodies, Insoluble cell fraction, Insoluble fraction, Ivanov, Laemmli sample buffer, Microbiol, Molecular mass, Nitrocellulose membrane, Other yeast proteases, Plasma membrane, Plasmid, Preliminary enrichment, Primer, Prion, Prion aggregation, Promoter, Protein, Pyg1, Pysg, Pysg bst1, Pysg8, Recombinant, Recombinant antigen, Recombinant protein, Recombinant proteins, Recombinant yeast, Saccharomyces, Saccharomyces cerevisiae, Same enzymes, Same time, Structural stability, Substantial share, Translational fusion, Trends genet, Uniform sites, Ura3, Ura3 leu2, Yeast, Yeast cells, Yeast producers, Yeast saccharomyces cerevisiae.
Abstract
Abstract: Effective expression of the HIV-1 core protein Gag-p55 was obtained in Saccharomyces cerevisiae under control of the inducible UASgal/CYC1 promoter as a translational fusion with the prion-forming NM domain of the translation terminator Sup35p (eRF3) of S. cerevisiae, where only poor expression of the original-type Gag-p55 was observed. A deletion within the Sup35NM prion-forming domain altering Sup35-associated [PSI] inheritance did not compromise expression of the Sup35NM Gag-p55 fusion protein. Therefore, either the mechanism of this phenomenon is not directly related to the effect of Sup35p prion-formation or the modified protein maintains residual prion-forming abilities. The recombinant Sup35p-Gag-p55 protein was quite stable under boiling in an alkali/sodium dodecyl sulfate (SDS) solution and completely retained its antigenic properties. Moreover, 10-min boiling of the native yeast cells in this solution allowed immediate inhibition of lysosomal and other yeast proteases, responsible for autolysis of many natural and recombinant proteins. The use of this method of preliminary enrichment for the recombinant fusion protein Sup35p-Gag-p55 with the SDS-alkaline extraction could be useful for yeast heterologous expression and purification of other of insoluble and unstable proteins. A translational fusion with the NM domain of Sup35p was also used to produce another poorly soluble protein, the L-chain of botulinum exotoxin A, in S. cerevisiae. When the Sup35p fragment was removed from the recombinant construct encoding a fused Sup35/BoNT protein, a dramatic drop in both transformation efficiency and growth rate of transformants was shown.
Url:
DOI: 10.1016/S0923-2508(00)01165-7
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Shevelev</name><affiliation wicri:level="3"><country xml:lang="fr">Russie</country><wicri:regionArea>Moscow AIDS Center, Moscow</wicri:regionArea><placeName><settlement type="city">Moscou</settlement><region>District fédéral central</region></placeName></affiliation></author><author><name sortKey="Fominov, Gleb V" sort="Fominov, Gleb V" uniqKey="Fominov G" first="Gleb V" last="Fominov">Gleb V. Fominov</name><affiliation wicri:level="1"><country xml:lang="fr">Russie</country><wicri:regionArea>V.M. Stepanov Laboratory of Protein Chemistry, GNIIgenetika, 1st Dorozhny pr., 1, 11345 Moscow</wicri:regionArea><placeName><settlement type="city">Moscou</settlement><region>District fédéral central</region></placeName></affiliation></author><author><name sortKey="Wojciechowska, Jana A" sort="Wojciechowska, Jana A" uniqKey="Wojciechowska J" first="Jana A" last="Wojciechowska">Jana A Wojciechowska</name><affiliation wicri:level="1"><country xml:lang="fr">Russie</country><wicri:regionArea>V.M. Stepanov Laboratory of Protein Chemistry, GNIIgenetika, 1st Dorozhny pr., 1, 11345 Moscow</wicri:regionArea><placeName><settlement type="city">Moscou</settlement><region>District fédéral central</region></placeName></affiliation></author><author><name sortKey="Asadi Mobarhan, Ali H" sort="Asadi Mobarhan, Ali H" uniqKey="Asadi Mobarhan A" first="Ali H" last="Asadi Mobarhan">Ali H. Asadi Mobarhan</name><affiliation wicri:level="3"><country xml:lang="fr">Russie</country><wicri:regionArea>Medical Center “Avicenna”, Moscow</wicri:regionArea><placeName><settlement type="city">Moscou</settlement><region>District fédéral central</region></placeName></affiliation></author><author><name sortKey="Vertiev, Yuri V" sort="Vertiev, Yuri V" uniqKey="Vertiev Y" first="Yuri V" last="Vertiev">Yuri V. 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Yankovsky</name><affiliation wicri:level="3"><country xml:lang="fr">Russie</country><wicri:regionArea>Bion-M, Moscow</wicri:regionArea><placeName><settlement type="city">Moscou</settlement><region>District fédéral central</region></placeName></affiliation><affiliation wicri:level="3"><country xml:lang="fr">Russie</country><wicri:regionArea>Genome Analysis Lab., Vavilov Institute of General Genetics RAS, Moscow</wicri:regionArea><placeName><settlement type="city">Moscou</settlement><region>District fédéral central</region></placeName></affiliation></author><author><name sortKey="Shevelev, A B" sort="Shevelev, A B" uniqKey="Shevelev A" first="A. B" last="Shevelev">A. B Shevelev</name><affiliation wicri:level="1"><country xml:lang="fr">Russie</country><wicri:regionArea>V.M. Stepanov Laboratory of Protein Chemistry, GNIIgenetika, 1st Dorozhny pr., 1, 11345 Moscow</wicri:regionArea><placeName><settlement type="city">Moscou</settlement><region>District fédéral central</region></placeName></affiliation><affiliation></affiliation><affiliation></affiliation></author></analytic><monogr></monogr><series><title level="j">Research in Microbiology</title><title level="j" type="abbrev">RESMIC</title><idno type="ISSN">0923-2508</idno><imprint><publisher>ELSEVIER</publisher><date type="published" when="2001">2001</date><biblScope unit="volume">152</biblScope><biblScope unit="issue">1</biblScope><biblScope unit="page" from="27">27</biblScope><biblScope unit="page" to="35">35</biblScope></imprint><idno type="ISSN">0923-2508</idno></series></biblStruct></sourceDesc><seriesStmt><idno type="ISSN">0923-2508</idno></seriesStmt></fileDesc><profileDesc><textClass><keywords scheme="KwdEn" xml:lang="en"><term>HIV</term><term>L-chain</term><term>Saccharomyces cerevisiae</term><term>botulinum toxin gene</term><term>heterologous expression</term><term>protein degradation</term><term>yeast prion</term></keywords><keywords scheme="Teeft" xml:lang="en"><term>Alkaline phosphatase</term><term>Amino acid residues</term><term>Bamhi</term><term>Bont</term><term>Botl</term><term>Botulinum neurotoxin</term><term>Bst1</term><term>Cerevisiae</term><term>Cerevisiae strain</term><term>Cerevisiae transformation</term><term>Chimeric</term><term>Chimeric gene</term><term>Chimeric protein</term><term>Cold spring harbor laboratory press</term><term>Coli</term><term>Derivative</term><term>Empty vector</term><term>Encoding</term><term>Escherichia coli</term><term>Fusion protein</term><term>Galactose induction</term><term>Gene</term><term>Gene encoding</term><term>Gene expression</term><term>Growth rate</term><term>Gure</term><term>Heterologous</term><term>Human virus type</term><term>Immunopositive product</term><term>Inclusion bodies</term><term>Insoluble cell fraction</term><term>Insoluble fraction</term><term>Ivanov</term><term>Laemmli sample buffer</term><term>Microbiol</term><term>Molecular mass</term><term>Nitrocellulose membrane</term><term>Other yeast proteases</term><term>Plasma membrane</term><term>Plasmid</term><term>Preliminary enrichment</term><term>Primer</term><term>Prion</term><term>Prion aggregation</term><term>Promoter</term><term>Protein</term><term>Pyg1</term><term>Pysg</term><term>Pysg bst1</term><term>Pysg8</term><term>Recombinant</term><term>Recombinant antigen</term><term>Recombinant protein</term><term>Recombinant proteins</term><term>Recombinant yeast</term><term>Saccharomyces</term><term>Saccharomyces cerevisiae</term><term>Same enzymes</term><term>Same time</term><term>Structural stability</term><term>Substantial share</term><term>Translational fusion</term><term>Trends genet</term><term>Uniform sites</term><term>Ura3</term><term>Ura3 leu2</term><term>Yeast</term><term>Yeast cells</term><term>Yeast producers</term><term>Yeast saccharomyces cerevisiae</term></keywords></textClass><langUsage><language ident="en">en</language></langUsage></profileDesc></teiHeader><front><div type="abstract" xml:lang="en">Abstract: Effective expression of the HIV-1 core protein Gag-p55 was obtained in Saccharomyces cerevisiae under control of the inducible UASgal/CYC1 promoter as a translational fusion with the prion-forming NM domain of the translation terminator Sup35p (eRF3) of S. cerevisiae, where only poor expression of the original-type Gag-p55 was observed. A deletion within the Sup35NM prion-forming domain altering Sup35-associated [PSI] inheritance did not compromise expression of the Sup35NM Gag-p55 fusion protein. Therefore, either the mechanism of this phenomenon is not directly related to the effect of Sup35p prion-formation or the modified protein maintains residual prion-forming abilities. The recombinant Sup35p-Gag-p55 protein was quite stable under boiling in an alkali/sodium dodecyl sulfate (SDS) solution and completely retained its antigenic properties. Moreover, 10-min boiling of the native yeast cells in this solution allowed immediate inhibition of lysosomal and other yeast proteases, responsible for autolysis of many natural and recombinant proteins. The use of this method of preliminary enrichment for the recombinant fusion protein Sup35p-Gag-p55 with the SDS-alkaline extraction could be useful for yeast heterologous expression and purification of other of insoluble and unstable proteins. A translational fusion with the NM domain of Sup35p was also used to produce another poorly soluble protein, the L-chain of botulinum exotoxin A, in S. cerevisiae. When the Sup35p fragment was removed from the recombinant construct encoding a fused Sup35/BoNT protein, a dramatic drop in both transformation efficiency and growth rate of transformants was shown.</div></front></TEI></record>Pour manipuler ce document sous Unix (Dilib)
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