Source:http://linkedlifedata.com/resource/pubmed/id/16213750
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| Predicate | Object |
|---|---|
| rdf:type | |
| lifeskim:mentions | |
| pubmed:issue |
2
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| pubmed:dateCreated |
2005-11-7
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| pubmed:abstractText |
Granulocyte-macrophage colony-stimulating factor (GM-CSF) regulates proliferation, differentiation, and function of hematopoietic progenitor cells. Aside from expansion of hematopoietic cells, GM-CSF has shown efficacy in other diseases, including Crohn's disease. While GM-CSF being clinically used in humans, the ability to perform mechanistic studies in murine models is difficult due to the limited availability and rapid clearance of murine GM-CSF in the peripheral blood. To address these issues, we efficiently expressed murine GM-CSF under the control of the AOX1 gene promoter in Pichia pastoris using the Mut(S) strain KM71H. We describe the unique conditions that are required for efficient production by high-density fermentation and purification of mGM-CSF protein. Recombinant mGM-CSF protein was purified by tangential flow ultrafiltration and preparative reverse phase chromatography. To address limited half life or rapid clearance in mice, recombinant murine GM-CSF was modified by lysine-directed polyethylene glycol conjugation (PEGylation). PEG-modified and unmodified proteins were characterized by amino terminus sequence analysis and matrix assisted laser desorption ionization time-of-flight mass spectrometry. Under the mild reaction conditions, the recombinant protein is efficiently modified by PEGylation on an average of 2-3 sites per molecule. In vivo treatment of mice with PEGylated mGM-CSF, but not the unmodified recombinant mGM-CSF, reproduces the potent colony stimulating effects of human GM-CSF in patients on myeloid progenitor populations, as assessed by FACs analysis. This simplified approach for the expression, purification, and modification of a biologically potent form of murine GM-CSF should facilitate the study of central mechanisms of action in murine disease models.
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| pubmed:language |
eng
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| pubmed:journal | |
| pubmed:citationSubset |
IM
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| pubmed:chemical |
http://linkedlifedata.com/resource/pubmed/chemical/Granulocyte-Macrophage...,
http://linkedlifedata.com/resource/pubmed/chemical/N-succinimidyl propionate,
http://linkedlifedata.com/resource/pubmed/chemical/Polyethylene Glycols,
http://linkedlifedata.com/resource/pubmed/chemical/Propionates,
http://linkedlifedata.com/resource/pubmed/chemical/Protein Sorting Signals,
http://linkedlifedata.com/resource/pubmed/chemical/Recombinant Proteins
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| pubmed:status |
MEDLINE
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| pubmed:month |
Dec
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| pubmed:issn |
1046-5928
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| pubmed:author |
pubmed-author:AnantShrikantS,
pubmed-author:BishnupuriKumar SKS,
pubmed-author:CrimminsDan LDL,
pubmed-author:DieckgraefeBrian KBK,
pubmed-author:HouchenCourtneyC,
pubmed-author:LiAnguoA,
pubmed-author:LinK JKJ,
pubmed-author:McDonaldKeely GKG,
pubmed-author:NewberryRodney DRD,
pubmed-author:SainathanSatheesh KSK,
pubmed-author:TuLiwenL
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| pubmed:issnType |
Print
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| pubmed:volume |
44
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| pubmed:owner |
NLM
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| pubmed:authorsComplete |
Y
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| pubmed:pagination |
94-103
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| pubmed:dateRevised |
2011-11-17
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| pubmed:meshHeading |
pubmed-meshheading:16213750-Amino Acid Sequence,
pubmed-meshheading:16213750-Animals,
pubmed-meshheading:16213750-Blotting, Western,
pubmed-meshheading:16213750-Fermentation,
pubmed-meshheading:16213750-Glycosylation,
pubmed-meshheading:16213750-Granulocyte-Macrophage Colony-Stimulating Factor,
pubmed-meshheading:16213750-Half-Life,
pubmed-meshheading:16213750-Male,
pubmed-meshheading:16213750-Metabolic Clearance Rate,
pubmed-meshheading:16213750-Mice,
pubmed-meshheading:16213750-Mice, Inbred C57BL,
pubmed-meshheading:16213750-Molecular Sequence Data,
pubmed-meshheading:16213750-Molecular Weight,
pubmed-meshheading:16213750-Pichia,
pubmed-meshheading:16213750-Polyethylene Glycols,
pubmed-meshheading:16213750-Propionates,
pubmed-meshheading:16213750-Protein Sorting Signals,
pubmed-meshheading:16213750-Recombinant Proteins,
pubmed-meshheading:16213750-Spectrometry, Mass, Matrix-Assisted Laser...
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| pubmed:year |
2005
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| pubmed:articleTitle |
PEGylated murine Granulocyte-macrophage colony-stimulating factor: production, purification, and characterization.
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| pubmed:affiliation |
Division of Gastroenterology, Department of Medicine, Washington University School of Medicine, Saint Louis, MO 63110, USA.
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| pubmed:publicationType |
Journal Article
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