Abstract：
: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.
journal_name
Protein Expr Purifjournal_title
Protein expression and purificationauthors
Sainathan SK,Tu L,Bishnupuri KS,Han M,Li A,Newberry RD,McDonald KG,Crimmins DL,Houchen C,Anant S,Dieckgraefe BKdoi
10.1016/j.pep.2005.08.014keywords:
subject
Has Abstractpub_date
2005-12-01 00:00:00pages
94-103issue
2eissn
1046-5928issn
1096-0279pii
S1046-5928(05)00295-0journal_volume
44pub_type
杂志文章abstract：:Ketol-acid reductoisomerase (EC 1.1.1.86) catalyzes the conversion of 2-aceto-2-hydroxyacids to 2-keto-3-hydroxyacids and their subsequent reduction by NADPH to 2,3-dihydroxyacids. The gene encoding the Escherichia coli enzyme was cloned and expressed as a hexahistidine-tagged fusion protein and the recombinant enzyme...
journal_title：Protein expression and purification
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journal_title：Protein expression and purification
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journal_title：Protein expression and purification
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journal_title：Protein expression and purification
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journal_title：Protein expression and purification
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journal_title：Protein expression and purification
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journal_title：Protein expression and purification
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journal_title：Protein expression and purification
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journal_title：Protein expression and purification
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doi：10.1016/s1046-5928(03)00046-9
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journal_title：Protein expression and purification
pub_type： 杂志文章
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更新日期：2000-04-01 00:00:00
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journal_title：Protein expression and purification
pub_type： 杂志文章
doi：10.1016/j.pep.2008.05.008
更新日期：2008-10-01 00:00:00
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journal_title：Protein expression and purification
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doi：10.1016/j.pep.2013.11.014
更新日期：2014-03-01 00:00:00
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journal_title：Protein expression and purification
pub_type： 杂志文章
doi：10.1006/prep.1996.0096
更新日期：1996-09-01 00:00:00
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journal_title：Protein expression and purification
pub_type： 杂志文章
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journal_title：Protein expression and purification
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doi：10.1016/j.pep.2011.12.001
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doi：10.1016/s1046-5928(03)00191-8
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journal_title：Protein expression and purification
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journal_title：Protein expression and purification
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journal_title：Protein expression and purification
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journal_title：Protein expression and purification
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journal_title：Protein expression and purification
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journal_title：Protein expression and purification
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journal_title：Protein expression and purification
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journal_title：Protein expression and purification
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journal_title：Protein expression and purification
pub_type： 杂志文章
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journal_title：Protein expression and purification
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journal_title：Protein expression and purification
pub_type： 杂志文章
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更新日期：2013-04-01 00:00:00
