title: PEGylated murine Granulocyte-macrophage colony-stimulating factor: production, purification, and characterization.
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.
authors.affiliation: Division of Gastroenterology, Department of Medicine, Washington University School of Medicine, Saint Louis, MO 63110, USA.
authors.forename: Satheesh K
authors.initials: SK
authors.lastname: Sainathan
authors.forename: Liwen
authors.initials: L
authors.lastname: Tu
authors.forename: Kumar S
authors.initials: KS
authors.lastname: Bishnupuri
authors.forename: Mei
authors.initials: M
authors.lastname: Han
authors.forename: Anguo
authors.initials: A
authors.lastname: Li
authors.forename: Rodney D
authors.initials: RD
authors.lastname: Newberry
authors.forename: Keely G
authors.initials: KG
authors.lastname: McDonald
authors.forename: Dan L
authors.initials: DL
authors.lastname: Crimmins
authors.forename: Courtney
authors.initials: C
authors.lastname: Houchen
authors.forename: Shrikant
authors.initials: S
authors.lastname: Anant
authors.forename: Brian K
authors.initials: BK
authors.lastname: Dieckgraefe
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D005285:Fermentation; D006031:Glycosylation; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000096:biosynthesis* / Q000737:chemistry / Q000493:pharmacokinetics; D006207:Half-Life; D008297:Male; D008657:Metabolic Clearance Rate; D051379:Mice; D008810:Mice, Inbred C57BL; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010843:Pichia / Q000235:genetics / Q000378:metabolism; D011092:Polyethylene Glycols / Q000737:chemistry* / Q000493:pharmacokinetics; D011422:Propionates / Q000737:chemistry; D021382:Protein Sorting Signals / Q000235:genetics; D011994:Recombinant Proteins; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
chemical_list: D011422:Propionates; D021382:Protein Sorting Signals; D011994:Recombinant Proteins; C021749:N-succinimidyl propionate; D011092:Polyethylene Glycols; D016178:Granulocyte-Macrophage Colony-Stimulating Factor