Purification of the M flax-rust resistance protein expressed in Pichia pastoris.
Identifieur interne : 000139 ( Main/Exploration ); précédent : 000138; suivant : 000140Purification of the M flax-rust resistance protein expressed in Pichia pastoris.
Auteurs : Simon A. Schmidt [Australie] ; Simon J. Williams ; Ching-I A. Wang ; Pradeep Sornaraj ; Ben James ; Bostjan Kobe ; Peter N. Dodds ; Jeffrey G. Ellis ; Peter A. AndersonSource :
- The Plant journal : for cell and molecular biology [ 0960-7412 ] ; 2007.
Descripteurs français
- KwdFr :
- Basidiomycota (MeSH), Expression des gènes (MeSH), Gènes de plante (MeSH), Lin (génétique), Lin (microbiologie), Maladies des plantes (génétique), Maladies des plantes (microbiologie), Pichia (génétique), Pichia (métabolisme), Plasmides (MeSH), Protéines recombinantes (biosynthèse), Protéines recombinantes (isolement et purification), Protéines végétales (biosynthèse), Protéines végétales (isolement et purification), Vecteurs génétiques (MeSH).
- MESH :
- biosynthèse : Protéines recombinantes, Protéines végétales.
- génétique : Lin, Maladies des plantes, Pichia.
- isolement et purification : Protéines recombinantes, Protéines végétales.
- microbiologie : Lin, Maladies des plantes.
- métabolisme : Pichia.
- Basidiomycota, Expression des gènes, Gènes de plante, Plasmides, Vecteurs génétiques.
English descriptors
- KwdEn :
- Basidiomycota (MeSH), Flax (genetics), Flax (microbiology), Gene Expression (MeSH), Genes, Plant (MeSH), Genetic Vectors (MeSH), Pichia (genetics), Pichia (metabolism), Plant Diseases (genetics), Plant Diseases (microbiology), Plant Proteins (biosynthesis), Plant Proteins (isolation & purification), Plasmids (MeSH), Recombinant Proteins (biosynthesis), Recombinant Proteins (isolation & purification).
- MESH :
- chemical , biosynthesis : Plant Proteins, Recombinant Proteins.
- genetics : Flax, Pichia, Plant Diseases.
- chemical , isolation & purification : Plant Proteins, Recombinant Proteins.
- metabolism : Pichia.
- microbiology : Flax, Plant Diseases.
- Basidiomycota, Gene Expression, Genes, Plant, Genetic Vectors, Plasmids.
Abstract
The M flax-rust resistance (R) gene is predicted to encode a 150-kDa protein of the Toll-interleukin-like receptor-nucleotide binding site-leucine rich repeat (TIR-NBS-LRR) class of plant disease resistance proteins and provides resistance against the Melampsora lini (flax rust) fungus carrying the AvrM avirulence gene. The extremely low level of this class of R proteins found in plant tissue has precluded their biochemical and structural analysis, and the study of these proteins has been largely restricted to genetic analyses and in vivo investigations. Here we report the production and purification of the M protein in the methalotrophic yeast, Pichia pastoris. Expression trials with five different constructs reveals optimum levels of soluble native M protein can be obtained as an N-terminally 9x His-tagged protein, in which the first 21 amino acids of the predicted wild-type protein are deleted. Expression was achieved using a high cell density fed-batch bioreactor culture at low temperature. M protein was purified to near homogeneity from whole-cell lysates using cation exchange, immobilised metal ion affinity chromatography and gel filtration with a final yield of approximately 3 mg of protein/1000 g wet weight of yeast cells lysed. The successful expression and purification of soluble M protein opens the way for biochemical and structural analysis of this class of important plant proteins.
DOI: 10.1111/j.1365-313X.2007.03104.x
PubMed: 17461785
Affiliations:
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Wang</name></author><author><name sortKey="Sornaraj, Pradeep" sort="Sornaraj, Pradeep" uniqKey="Sornaraj P" first="Pradeep" last="Sornaraj">Pradeep Sornaraj</name></author><author><name sortKey="James, Ben" sort="James, Ben" uniqKey="James B" first="Ben" last="James">Ben James</name></author><author><name sortKey="Kobe, Bostjan" sort="Kobe, Bostjan" uniqKey="Kobe B" first="Bostjan" last="Kobe">Bostjan Kobe</name></author><author><name sortKey="Dodds, Peter N" sort="Dodds, Peter N" uniqKey="Dodds P" first="Peter N" last="Dodds">Peter N. Dodds</name></author><author><name sortKey="Ellis, Jeffrey G" sort="Ellis, Jeffrey G" uniqKey="Ellis J" first="Jeffrey G" last="Ellis">Jeffrey G. Ellis</name></author><author><name sortKey="Anderson, Peter A" sort="Anderson, Peter A" uniqKey="Anderson P" first="Peter A" last="Anderson">Peter A. 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Schmidt</name><affiliation wicri:level="1"><nlm:affiliation>The School of Biological Sciences, Flinders University, G.P.O. Box 2100, Adelaide 5001, Australia.</nlm:affiliation><country xml:lang="fr">Australie</country><wicri:regionArea>The School of Biological Sciences, Flinders University, G.P.O. Box 2100, Adelaide 5001</wicri:regionArea><wicri:noRegion>Adelaide 5001</wicri:noRegion></affiliation></author><author><name sortKey="Williams, Simon J" sort="Williams, Simon J" uniqKey="Williams S" first="Simon J" last="Williams">Simon J. Williams</name></author><author><name sortKey="Wang, Ching I A" sort="Wang, Ching I A" uniqKey="Wang C" first="Ching-I A" last="Wang">Ching-I A. Wang</name></author><author><name sortKey="Sornaraj, Pradeep" sort="Sornaraj, Pradeep" uniqKey="Sornaraj P" first="Pradeep" last="Sornaraj">Pradeep Sornaraj</name></author><author><name sortKey="James, Ben" sort="James, Ben" uniqKey="James B" first="Ben" last="James">Ben James</name></author><author><name sortKey="Kobe, Bostjan" sort="Kobe, Bostjan" uniqKey="Kobe B" first="Bostjan" last="Kobe">Bostjan Kobe</name></author><author><name sortKey="Dodds, Peter N" sort="Dodds, Peter N" uniqKey="Dodds P" first="Peter N" last="Dodds">Peter N. Dodds</name></author><author><name sortKey="Ellis, Jeffrey G" sort="Ellis, Jeffrey G" uniqKey="Ellis J" first="Jeffrey G" last="Ellis">Jeffrey G. Ellis</name></author><author><name sortKey="Anderson, Peter A" sort="Anderson, Peter A" uniqKey="Anderson P" first="Peter A" last="Anderson">Peter A. Anderson</name></author></analytic><series><title level="j">The Plant journal : for cell and molecular biology</title><idno type="ISSN">0960-7412</idno><imprint><date when="2007" type="published">2007</date></imprint></series></biblStruct></sourceDesc></fileDesc><profileDesc><textClass><keywords scheme="KwdEn" xml:lang="en"><term>Basidiomycota (MeSH)</term><term>Flax (genetics)</term><term>Flax (microbiology)</term><term>Gene Expression (MeSH)</term><term>Genes, Plant (MeSH)</term><term>Genetic Vectors (MeSH)</term><term>Pichia (genetics)</term><term>Pichia (metabolism)</term><term>Plant Diseases (genetics)</term><term>Plant Diseases (microbiology)</term><term>Plant Proteins (biosynthesis)</term><term>Plant Proteins (isolation & purification)</term><term>Plasmids (MeSH)</term><term>Recombinant Proteins (biosynthesis)</term><term>Recombinant Proteins (isolation & purification)</term></keywords><keywords scheme="KwdFr" xml:lang="fr"><term>Basidiomycota (MeSH)</term><term>Expression des gènes (MeSH)</term><term>Gènes de plante (MeSH)</term><term>Lin (génétique)</term><term>Lin (microbiologie)</term><term>Maladies des plantes (génétique)</term><term>Maladies des plantes (microbiologie)</term><term>Pichia (génétique)</term><term>Pichia (métabolisme)</term><term>Plasmides (MeSH)</term><term>Protéines recombinantes (biosynthèse)</term><term>Protéines recombinantes (isolement et purification)</term><term>Protéines végétales (biosynthèse)</term><term>Protéines végétales (isolement et purification)</term><term>Vecteurs génétiques (MeSH)</term></keywords><keywords scheme="MESH" type="chemical" qualifier="biosynthesis" xml:lang="en"><term>Plant Proteins</term><term>Recombinant Proteins</term></keywords><keywords scheme="MESH" qualifier="biosynthèse" xml:lang="fr"><term>Protéines recombinantes</term><term>Protéines végétales</term></keywords><keywords scheme="MESH" qualifier="genetics" xml:lang="en"><term>Flax</term><term>Pichia</term><term>Plant Diseases</term></keywords><keywords scheme="MESH" qualifier="génétique" xml:lang="fr"><term>Lin</term><term>Maladies des plantes</term><term>Pichia</term></keywords><keywords scheme="MESH" type="chemical" qualifier="isolation & purification" xml:lang="en"><term>Plant Proteins</term><term>Recombinant Proteins</term></keywords><keywords scheme="MESH" qualifier="isolement et purification" xml:lang="fr"><term>Protéines recombinantes</term><term>Protéines végétales</term></keywords><keywords scheme="MESH" qualifier="metabolism" xml:lang="en"><term>Pichia</term></keywords><keywords scheme="MESH" qualifier="microbiologie" xml:lang="fr"><term>Lin</term><term>Maladies des plantes</term></keywords><keywords scheme="MESH" qualifier="microbiology" xml:lang="en"><term>Flax</term><term>Plant Diseases</term></keywords><keywords scheme="MESH" qualifier="métabolisme" xml:lang="fr"><term>Pichia</term></keywords><keywords scheme="MESH" xml:lang="en"><term>Basidiomycota</term><term>Gene Expression</term><term>Genes, Plant</term><term>Genetic Vectors</term><term>Plasmids</term></keywords><keywords scheme="MESH" xml:lang="fr"><term>Basidiomycota</term><term>Expression des gènes</term><term>Gènes de plante</term><term>Plasmides</term><term>Vecteurs génétiques</term></keywords></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en">The M flax-rust resistance (R) gene is predicted to encode a 150-kDa protein of the Toll-interleukin-like receptor-nucleotide binding site-leucine rich repeat (TIR-NBS-LRR) class of plant disease resistance proteins and provides resistance against the Melampsora lini (flax rust) fungus carrying the AvrM avirulence gene. The extremely low level of this class of R proteins found in plant tissue has precluded their biochemical and structural analysis, and the study of these proteins has been largely restricted to genetic analyses and in vivo investigations. Here we report the production and purification of the M protein in the methalotrophic yeast, Pichia pastoris. Expression trials with five different constructs reveals optimum levels of soluble native M protein can be obtained as an N-terminally 9x His-tagged protein, in which the first 21 amino acids of the predicted wild-type protein are deleted. Expression was achieved using a high cell density fed-batch bioreactor culture at low temperature. M protein was purified to near homogeneity from whole-cell lysates using cation exchange, immobilised metal ion affinity chromatography and gel filtration with a final yield of approximately 3 mg of protein/1000 g wet weight of yeast cells lysed. The successful expression and purification of soluble M protein opens the way for biochemical and structural analysis of this class of important plant proteins.</div></front></TEI><pubmed><MedlineCitation Status="MEDLINE" Owner="NLM"><PMID Version="1">17461785</PMID><DateCompleted><Year>2007</Year><Month>09</Month><Day>05</Day></DateCompleted><DateRevised><Year>2007</Year><Month>06</Month><Day>13</Day></DateRevised><Article PubModel="Print-Electronic"><Journal><ISSN IssnType="Print">0960-7412</ISSN><JournalIssue CitedMedium="Print"><Volume>50</Volume><Issue>6</Issue><PubDate><Year>2007</Year><Month>Jun</Month></PubDate></JournalIssue><Title>The Plant journal : for cell and molecular biology</Title><ISOAbbreviation>Plant J</ISOAbbreviation></Journal><ArticleTitle>Purification of the M flax-rust resistance protein expressed in Pichia pastoris.</ArticleTitle><Pagination><MedlinePgn>1107-17</MedlinePgn></Pagination><Abstract><AbstractText>The M flax-rust resistance (R) gene is predicted to encode a 150-kDa protein of the Toll-interleukin-like receptor-nucleotide binding site-leucine rich repeat (TIR-NBS-LRR) class of plant disease resistance proteins and provides resistance against the Melampsora lini (flax rust) fungus carrying the AvrM avirulence gene. The extremely low level of this class of R proteins found in plant tissue has precluded their biochemical and structural analysis, and the study of these proteins has been largely restricted to genetic analyses and in vivo investigations. Here we report the production and purification of the M protein in the methalotrophic yeast, Pichia pastoris. Expression trials with five different constructs reveals optimum levels of soluble native M protein can be obtained as an N-terminally 9x His-tagged protein, in which the first 21 amino acids of the predicted wild-type protein are deleted. Expression was achieved using a high cell density fed-batch bioreactor culture at low temperature. M protein was purified to near homogeneity from whole-cell lysates using cation exchange, immobilised metal ion affinity chromatography and gel filtration with a final yield of approximately 3 mg of protein/1000 g wet weight of yeast cells lysed. The successful expression and purification of soluble M protein opens the way for biochemical and structural analysis of this class of important plant proteins.</AbstractText></Abstract><AuthorList CompleteYN="Y"><Author ValidYN="Y"><LastName>Schmidt</LastName><ForeName>Simon A</ForeName><Initials>SA</Initials><AffiliationInfo><Affiliation>The School of Biological Sciences, Flinders University, G.P.O. Box 2100, Adelaide 5001, Australia.</Affiliation></AffiliationInfo></Author><Author ValidYN="Y"><LastName>Williams</LastName><ForeName>Simon J</ForeName><Initials>SJ</Initials></Author><Author ValidYN="Y"><LastName>Wang</LastName><ForeName>Ching-I A</ForeName><Initials>CI</Initials></Author><Author ValidYN="Y"><LastName>Sornaraj</LastName><ForeName>Pradeep</ForeName><Initials>P</Initials></Author><Author ValidYN="Y"><LastName>James</LastName><ForeName>Ben</ForeName><Initials>B</Initials></Author><Author ValidYN="Y"><LastName>Kobe</LastName><ForeName>Bostjan</ForeName><Initials>B</Initials></Author><Author ValidYN="Y"><LastName>Dodds</LastName><ForeName>Peter N</ForeName><Initials>PN</Initials></Author><Author ValidYN="Y"><LastName>Ellis</LastName><ForeName>Jeffrey G</ForeName><Initials>JG</Initials></Author><Author ValidYN="Y"><LastName>Anderson</LastName><ForeName>Peter A</ForeName><Initials>PA</Initials></Author></AuthorList><Language>eng</Language><PublicationTypeList><PublicationType UI="D016428">Journal Article</PublicationType><PublicationType UI="D013485">Research Support, Non-U.S. Gov't</PublicationType></PublicationTypeList><ArticleDate DateType="Electronic"><Year>2007</Year><Month>04</Month><Day>25</Day></ArticleDate></Article><MedlineJournalInfo><Country>England</Country><MedlineTA>Plant J</MedlineTA><NlmUniqueID>9207397</NlmUniqueID><ISSNLinking>0960-7412</ISSNLinking></MedlineJournalInfo><ChemicalList><Chemical><RegistryNumber>0</RegistryNumber><NameOfSubstance UI="D010940">Plant Proteins</NameOfSubstance></Chemical><Chemical><RegistryNumber>0</RegistryNumber><NameOfSubstance UI="D011994">Recombinant Proteins</NameOfSubstance></Chemical></ChemicalList><CitationSubset>IM</CitationSubset><MeshHeadingList><MeshHeading><DescriptorName UI="D001487" MajorTopicYN="N">Basidiomycota</DescriptorName></MeshHeading><MeshHeading><DescriptorName UI="D019597" MajorTopicYN="N">Flax</DescriptorName><QualifierName UI="Q000235" MajorTopicYN="Y">genetics</QualifierName><QualifierName UI="Q000382" MajorTopicYN="N">microbiology</QualifierName></MeshHeading><MeshHeading><DescriptorName UI="D015870" MajorTopicYN="N">Gene Expression</DescriptorName></MeshHeading><MeshHeading><DescriptorName UI="D017343" MajorTopicYN="N">Genes, Plant</DescriptorName></MeshHeading><MeshHeading><DescriptorName UI="D005822" MajorTopicYN="N">Genetic Vectors</DescriptorName></MeshHeading><MeshHeading><DescriptorName UI="D010843" MajorTopicYN="N">Pichia</DescriptorName><QualifierName UI="Q000235" MajorTopicYN="N">genetics</QualifierName><QualifierName UI="Q000378" MajorTopicYN="Y">metabolism</QualifierName></MeshHeading><MeshHeading><DescriptorName UI="D010935" MajorTopicYN="N">Plant Diseases</DescriptorName><QualifierName UI="Q000235" MajorTopicYN="N">genetics</QualifierName><QualifierName UI="Q000382" MajorTopicYN="N">microbiology</QualifierName></MeshHeading><MeshHeading><DescriptorName UI="D010940" MajorTopicYN="N">Plant Proteins</DescriptorName><QualifierName UI="Q000096" MajorTopicYN="N">biosynthesis</QualifierName><QualifierName UI="Q000302" MajorTopicYN="Y">isolation & purification</QualifierName></MeshHeading><MeshHeading><DescriptorName UI="D010957" MajorTopicYN="N">Plasmids</DescriptorName></MeshHeading><MeshHeading><DescriptorName UI="D011994" MajorTopicYN="N">Recombinant Proteins</DescriptorName><QualifierName UI="Q000096" MajorTopicYN="N">biosynthesis</QualifierName><QualifierName UI="Q000302" MajorTopicYN="N">isolation & purification</QualifierName></MeshHeading></MeshHeadingList></MedlineCitation><PubmedData><History><PubMedPubDate PubStatus="pubmed"><Year>2007</Year><Month>4</Month><Day>28</Day><Hour>9</Hour><Minute>0</Minute></PubMedPubDate><PubMedPubDate PubStatus="medline"><Year>2007</Year><Month>9</Month><Day>6</Day><Hour>9</Hour><Minute>0</Minute></PubMedPubDate><PubMedPubDate PubStatus="entrez"><Year>2007</Year><Month>4</Month><Day>28</Day><Hour>9</Hour><Minute>0</Minute></PubMedPubDate></History><PublicationStatus>ppublish</PublicationStatus><ArticleIdList><ArticleId IdType="pubmed">17461785</ArticleId><ArticleId IdType="pii">TPJ3104</ArticleId><ArticleId IdType="doi">10.1111/j.1365-313X.2007.03104.x</ArticleId></ArticleIdList></PubmedData></pubmed><affiliations><list><country><li>Australie</li></country></list><tree><noCountry><name sortKey="Anderson, Peter A" sort="Anderson, Peter A" uniqKey="Anderson P" first="Peter A" last="Anderson">Peter A. Anderson</name><name sortKey="Dodds, Peter N" sort="Dodds, Peter N" uniqKey="Dodds P" first="Peter N" last="Dodds">Peter N. Dodds</name><name sortKey="Ellis, Jeffrey G" sort="Ellis, Jeffrey G" uniqKey="Ellis J" first="Jeffrey G" last="Ellis">Jeffrey G. Ellis</name><name sortKey="James, Ben" sort="James, Ben" uniqKey="James B" first="Ben" last="James">Ben James</name><name sortKey="Kobe, Bostjan" sort="Kobe, Bostjan" uniqKey="Kobe B" first="Bostjan" last="Kobe">Bostjan Kobe</name><name sortKey="Sornaraj, Pradeep" sort="Sornaraj, Pradeep" uniqKey="Sornaraj P" first="Pradeep" last="Sornaraj">Pradeep Sornaraj</name><name sortKey="Wang, Ching I A" sort="Wang, Ching I A" uniqKey="Wang C" first="Ching-I A" last="Wang">Ching-I A. Wang</name><name sortKey="Williams, Simon J" sort="Williams, Simon J" uniqKey="Williams S" first="Simon J" last="Williams">Simon J. Williams</name></noCountry><country name="Australie"><noRegion><name sortKey="Schmidt, Simon A" sort="Schmidt, Simon A" uniqKey="Schmidt S" first="Simon A" last="Schmidt">Simon A. Schmidt</name></noRegion></country></tree></affiliations></record>Pour manipuler ce document sous Unix (Dilib)
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