PEGylated murine Granulocyte-macrophage colony-stimulating factor: production, purification, and characterization

Protein Expr Purif. 2005 Dec;44(2):94-103. doi: 10.1016/j.pep.2005.08.014. Epub 2005 Sep 20.


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.

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Blotting, Western
  • Fermentation
  • Glycosylation
  • Granulocyte-Macrophage Colony-Stimulating Factor / biosynthesis*
  • Granulocyte-Macrophage Colony-Stimulating Factor / chemistry
  • Granulocyte-Macrophage Colony-Stimulating Factor / pharmacokinetics
  • Half-Life
  • Male
  • Metabolic Clearance Rate
  • Mice
  • Mice, Inbred C57BL
  • Molecular Sequence Data
  • Molecular Weight
  • Pichia / genetics
  • Pichia / metabolism
  • Polyethylene Glycols / chemistry*
  • Polyethylene Glycols / pharmacokinetics
  • Propionates / chemistry
  • Protein Sorting Signals / genetics
  • Recombinant Proteins
  • Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization


  • Propionates
  • Protein Sorting Signals
  • Recombinant Proteins
  • N-succinimidyl propionate
  • Polyethylene Glycols
  • Granulocyte-Macrophage Colony-Stimulating Factor