Preparation of human vascular endothelial growth factor-D for structural and preclinical therapeutic studies.
Identifieur interne : 003F99 ( Main/Exploration ); précédent : 003F98; suivant : 004000Preparation of human vascular endothelial growth factor-D for structural and preclinical therapeutic studies.
Auteurs : Natalia Davydova [Australie] ; Victor A. Streltsov ; Sally Roufail ; George O. Lovrecz ; Steven A. Stacker ; Timothy E. Adams ; Marc G. AchenSource :
- Protein expression and purification [ 1096-0279 ] ; 2012.
Descripteurs français
- KwdFr :
- Cristallisation, Expression des gènes, Facteur de croissance endothéliale vasculaire de type D (), Facteur de croissance endothéliale vasculaire de type D (génétique), Facteur de croissance endothéliale vasculaire de type D (isolement et purification), Glycosylation, Humains, Liaison aux protéines, Lignée cellulaire, Multimérisation de protéines, Mutation, Structure tertiaire des protéines, Vecteurs génétiques (génétique).
- MESH :
- génétique : Facteur de croissance endothéliale vasculaire de type D, Vecteurs génétiques.
- isolement et purification : Facteur de croissance endothéliale vasculaire de type D.
- Cristallisation, Expression des gènes, Facteur de croissance endothéliale vasculaire de type D, Glycosylation, Humains, Liaison aux protéines, Lignée cellulaire, Multimérisation de protéines, Mutation, Structure tertiaire des protéines.
English descriptors
- KwdEn :
- Cell Line, Crystallization, Gene Expression, Genetic Vectors (genetics), Glycosylation, Humans, Mutation, Protein Binding, Protein Multimerization, Protein Structure, Tertiary, Vascular Endothelial Growth Factor D (chemistry), Vascular Endothelial Growth Factor D (genetics), Vascular Endothelial Growth Factor D (isolation & purification).
- MESH :
- chemical , chemistry : Vascular Endothelial Growth Factor D.
- genetics : Genetic Vectors, Vascular Endothelial Growth Factor D.
- chemical , isolation & purification : Vascular Endothelial Growth Factor D.
- Cell Line, Crystallization, Gene Expression, Glycosylation, Humans, Mutation, Protein Binding, Protein Multimerization, Protein Structure, Tertiary.
Abstract
Vascular endothelial growth factor-D (VEGF-D), a secreted angiogenic and lymphangiogenic glycoprotein, enhances tumor growth and metastasis in animal models, and its expression correlates with metastasis and poor patient outcome in some cancers - it is therefore considered a target for novel anti-cancer therapeutics. The definition of the structure of the complex of VEGF-D bound to its receptors would be beneficial for design of inhibitors of VEGF-D signaling aimed at restricting the growth and spread of cancer. In addition, there is interest in using VEGF-D protein for therapeutic angiogenesis and lymphangiogenesis in the settings of cardiovascular diseases and lymphedema, respectively. However, VEGF-D has proven difficult to express and purify in a highly bioactive form due to a tendency to exist as monomers rather than bioactive dimers. Here we describe a protocol for expression and purification of mature human VEGF-D, and a mutant thereof with reduced glycosylation, potentially suitable for preclinical therapeutic and structural studies, respectively. The degree of glycosylation in mature VEGF-D was reduced by eliminating one of the two N-glycosylation sites, and expressing the protein in Lec3.2.8.1 cells which had reduced glycosylation capacity. Mature VEGF-D and the glycosylation mutant were each enriched for the biologically active dimeric form by optimizing the separation of dimer from monomer via gel filtration, followed by conversion of remaining monomers to dimers via treatment with cysteine. The glycosylation mutant of VEGF-D intended for structural studies preserved all the cysteine residues of mature VEGF-D, in contrast to previous structural studies, exhibited comparable receptor binding to mature VEGF-D and might facilitate structural studies of the VEGF-D/VEGFR-3 complex.
DOI: 10.1016/j.pep.2012.01.001
PubMed: 22261343
Affiliations:
Links toward previous steps (curation, corpus...)
- to stream PubMed, to step Corpus: 002307
- to stream PubMed, to step Curation: 002307
- to stream PubMed, to step Checkpoint: 002307
- to stream Ncbi, to step Merge: 004993
- to stream Ncbi, to step Curation: 004993
- to stream Ncbi, to step Checkpoint: 004993
- to stream Main, to step Merge: 004013
- to stream Main, to step Curation: 003F99
Le document en format XML
<record><TEI><teiHeader><fileDesc><titleStmt><title xml:lang="en">Preparation of human vascular endothelial growth factor-D for structural and preclinical therapeutic studies.</title><author><name sortKey="Davydova, Natalia" sort="Davydova, Natalia" uniqKey="Davydova N" first="Natalia" last="Davydova">Natalia Davydova</name><affiliation wicri:level="1"><nlm:affiliation>Ludwig Institute for Cancer Research, P.O. Box 2008, Royal Melbourne Hospital, Victoria 3050, Australia.</nlm:affiliation><country xml:lang="fr">Australie</country><wicri:regionArea>Ludwig Institute for Cancer Research, P.O. Box 2008, Royal Melbourne Hospital, Victoria 3050</wicri:regionArea><wicri:noRegion>Victoria 3050</wicri:noRegion></affiliation></author><author><name sortKey="Streltsov, Victor A" sort="Streltsov, Victor A" uniqKey="Streltsov V" first="Victor A" last="Streltsov">Victor A. Streltsov</name></author><author><name sortKey="Roufail, Sally" sort="Roufail, Sally" uniqKey="Roufail S" first="Sally" last="Roufail">Sally Roufail</name></author><author><name sortKey="Lovrecz, George O" sort="Lovrecz, George O" uniqKey="Lovrecz G" first="George O" last="Lovrecz">George O. Lovrecz</name></author><author><name sortKey="Stacker, Steven A" sort="Stacker, Steven A" uniqKey="Stacker S" first="Steven A" last="Stacker">Steven A. Stacker</name></author><author><name sortKey="Adams, Timothy E" sort="Adams, Timothy E" uniqKey="Adams T" first="Timothy E" last="Adams">Timothy E. Adams</name></author><author><name sortKey="Achen, Marc G" sort="Achen, Marc G" uniqKey="Achen M" first="Marc G" last="Achen">Marc G. Achen</name></author></titleStmt><publicationStmt><idno type="wicri:source">PubMed</idno><date when="2012">2012</date><idno type="RBID">pubmed:22261343</idno><idno type="pmid">22261343</idno><idno type="doi">10.1016/j.pep.2012.01.001</idno><idno type="wicri:Area/PubMed/Corpus">002307</idno><idno type="wicri:explorRef" wicri:stream="PubMed" wicri:step="Corpus" wicri:corpus="PubMed">002307</idno><idno type="wicri:Area/PubMed/Curation">002307</idno><idno type="wicri:explorRef" wicri:stream="PubMed" wicri:step="Curation">002307</idno><idno type="wicri:Area/PubMed/Checkpoint">002307</idno><idno type="wicri:explorRef" wicri:stream="Checkpoint" wicri:step="PubMed">002307</idno><idno type="wicri:Area/Ncbi/Merge">004993</idno><idno type="wicri:Area/Ncbi/Curation">004993</idno><idno type="wicri:Area/Ncbi/Checkpoint">004993</idno><idno type="wicri:Area/Main/Merge">004013</idno><idno type="wicri:Area/Main/Curation">003F99</idno><idno type="wicri:Area/Main/Exploration">003F99</idno></publicationStmt><sourceDesc><biblStruct><analytic><title xml:lang="en">Preparation of human vascular endothelial growth factor-D for structural and preclinical therapeutic studies.</title><author><name sortKey="Davydova, Natalia" sort="Davydova, Natalia" uniqKey="Davydova N" first="Natalia" last="Davydova">Natalia Davydova</name><affiliation wicri:level="1"><nlm:affiliation>Ludwig Institute for Cancer Research, P.O. Box 2008, Royal Melbourne Hospital, Victoria 3050, Australia.</nlm:affiliation><country xml:lang="fr">Australie</country><wicri:regionArea>Ludwig Institute for Cancer Research, P.O. Box 2008, Royal Melbourne Hospital, Victoria 3050</wicri:regionArea><wicri:noRegion>Victoria 3050</wicri:noRegion></affiliation></author><author><name sortKey="Streltsov, Victor A" sort="Streltsov, Victor A" uniqKey="Streltsov V" first="Victor A" last="Streltsov">Victor A. Streltsov</name></author><author><name sortKey="Roufail, Sally" sort="Roufail, Sally" uniqKey="Roufail S" first="Sally" last="Roufail">Sally Roufail</name></author><author><name sortKey="Lovrecz, George O" sort="Lovrecz, George O" uniqKey="Lovrecz G" first="George O" last="Lovrecz">George O. Lovrecz</name></author><author><name sortKey="Stacker, Steven A" sort="Stacker, Steven A" uniqKey="Stacker S" first="Steven A" last="Stacker">Steven A. Stacker</name></author><author><name sortKey="Adams, Timothy E" sort="Adams, Timothy E" uniqKey="Adams T" first="Timothy E" last="Adams">Timothy E. Adams</name></author><author><name sortKey="Achen, Marc G" sort="Achen, Marc G" uniqKey="Achen M" first="Marc G" last="Achen">Marc G. Achen</name></author></analytic><series><title level="j">Protein expression and purification</title><idno type="eISSN">1096-0279</idno><imprint><date when="2012" type="published">2012</date></imprint></series></biblStruct></sourceDesc></fileDesc><profileDesc><textClass><keywords scheme="KwdEn" xml:lang="en"><term>Cell Line</term><term>Crystallization</term><term>Gene Expression</term><term>Genetic Vectors (genetics)</term><term>Glycosylation</term><term>Humans</term><term>Mutation</term><term>Protein Binding</term><term>Protein Multimerization</term><term>Protein Structure, Tertiary</term><term>Vascular Endothelial Growth Factor D (chemistry)</term><term>Vascular Endothelial Growth Factor D (genetics)</term><term>Vascular Endothelial Growth Factor D (isolation & purification)</term></keywords><keywords scheme="KwdFr" xml:lang="fr"><term>Cristallisation</term><term>Expression des gènes</term><term>Facteur de croissance endothéliale vasculaire de type D ()</term><term>Facteur de croissance endothéliale vasculaire de type D (génétique)</term><term>Facteur de croissance endothéliale vasculaire de type D (isolement et purification)</term><term>Glycosylation</term><term>Humains</term><term>Liaison aux protéines</term><term>Lignée cellulaire</term><term>Multimérisation de protéines</term><term>Mutation</term><term>Structure tertiaire des protéines</term><term>Vecteurs génétiques (génétique)</term></keywords><keywords scheme="MESH" type="chemical" qualifier="chemistry" xml:lang="en"><term>Vascular Endothelial Growth Factor D</term></keywords><keywords scheme="MESH" qualifier="genetics" xml:lang="en"><term>Genetic Vectors</term><term>Vascular Endothelial Growth Factor D</term></keywords><keywords scheme="MESH" qualifier="génétique" xml:lang="fr"><term>Facteur de croissance endothéliale vasculaire de type D</term><term>Vecteurs génétiques</term></keywords><keywords scheme="MESH" type="chemical" qualifier="isolation & purification" xml:lang="en"><term>Vascular Endothelial Growth Factor D</term></keywords><keywords scheme="MESH" qualifier="isolement et purification" xml:lang="fr"><term>Facteur de croissance endothéliale vasculaire de type D</term></keywords><keywords scheme="MESH" xml:lang="en"><term>Cell Line</term><term>Crystallization</term><term>Gene Expression</term><term>Glycosylation</term><term>Humans</term><term>Mutation</term><term>Protein Binding</term><term>Protein Multimerization</term><term>Protein Structure, Tertiary</term></keywords><keywords scheme="MESH" xml:lang="fr"><term>Cristallisation</term><term>Expression des gènes</term><term>Facteur de croissance endothéliale vasculaire de type D</term><term>Glycosylation</term><term>Humains</term><term>Liaison aux protéines</term><term>Lignée cellulaire</term><term>Multimérisation de protéines</term><term>Mutation</term><term>Structure tertiaire des protéines</term></keywords></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en">Vascular endothelial growth factor-D (VEGF-D), a secreted angiogenic and lymphangiogenic glycoprotein, enhances tumor growth and metastasis in animal models, and its expression correlates with metastasis and poor patient outcome in some cancers - it is therefore considered a target for novel anti-cancer therapeutics. The definition of the structure of the complex of VEGF-D bound to its receptors would be beneficial for design of inhibitors of VEGF-D signaling aimed at restricting the growth and spread of cancer. In addition, there is interest in using VEGF-D protein for therapeutic angiogenesis and lymphangiogenesis in the settings of cardiovascular diseases and lymphedema, respectively. However, VEGF-D has proven difficult to express and purify in a highly bioactive form due to a tendency to exist as monomers rather than bioactive dimers. Here we describe a protocol for expression and purification of mature human VEGF-D, and a mutant thereof with reduced glycosylation, potentially suitable for preclinical therapeutic and structural studies, respectively. The degree of glycosylation in mature VEGF-D was reduced by eliminating one of the two N-glycosylation sites, and expressing the protein in Lec3.2.8.1 cells which had reduced glycosylation capacity. Mature VEGF-D and the glycosylation mutant were each enriched for the biologically active dimeric form by optimizing the separation of dimer from monomer via gel filtration, followed by conversion of remaining monomers to dimers via treatment with cysteine. The glycosylation mutant of VEGF-D intended for structural studies preserved all the cysteine residues of mature VEGF-D, in contrast to previous structural studies, exhibited comparable receptor binding to mature VEGF-D and might facilitate structural studies of the VEGF-D/VEGFR-3 complex.</div></front></TEI><affiliations><list><country><li>Australie</li></country></list><tree><noCountry><name sortKey="Achen, Marc G" sort="Achen, Marc G" uniqKey="Achen M" first="Marc G" last="Achen">Marc G. Achen</name><name sortKey="Adams, Timothy E" sort="Adams, Timothy E" uniqKey="Adams T" first="Timothy E" last="Adams">Timothy E. Adams</name><name sortKey="Lovrecz, George O" sort="Lovrecz, George O" uniqKey="Lovrecz G" first="George O" last="Lovrecz">George O. Lovrecz</name><name sortKey="Roufail, Sally" sort="Roufail, Sally" uniqKey="Roufail S" first="Sally" last="Roufail">Sally Roufail</name><name sortKey="Stacker, Steven A" sort="Stacker, Steven A" uniqKey="Stacker S" first="Steven A" last="Stacker">Steven A. Stacker</name><name sortKey="Streltsov, Victor A" sort="Streltsov, Victor A" uniqKey="Streltsov V" first="Victor A" last="Streltsov">Victor A. Streltsov</name></noCountry><country name="Australie"><noRegion><name sortKey="Davydova, Natalia" sort="Davydova, Natalia" uniqKey="Davydova N" first="Natalia" last="Davydova">Natalia Davydova</name></noRegion></country></tree></affiliations></record>Pour manipuler ce document sous Unix (Dilib)
EXPLOR_STEP=$WICRI_ROOT/Wicri/Sante/explor/LymphedemaV1/Data/Main/Exploration
HfdSelect -h $EXPLOR_STEP/biblio.hfd -nk 003F99 | SxmlIndent | more
Ou
HfdSelect -h $EXPLOR_AREA/Data/Main/Exploration/biblio.hfd -nk 003F99 | SxmlIndent | more
Pour mettre un lien sur cette page dans le réseau Wicri
{{Explor lien
|wiki= Wicri/Sante
|area= LymphedemaV1
|flux= Main
|étape= Exploration
|type= RBID
|clé= pubmed:22261343
|texte= Preparation of human vascular endothelial growth factor-D for structural and preclinical therapeutic studies.
}}
Pour générer des pages wiki
HfdIndexSelect -h $EXPLOR_AREA/Data/Main/Exploration/RBID.i -Sk "pubmed:22261343" \
| HfdSelect -Kh $EXPLOR_AREA/Data/Main/Exploration/biblio.hfd \
| NlmPubMed2Wicri -a LymphedemaV1
| This area was generated with Dilib version V0.6.31. |  |