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

Satheesh K. Sainathan, Liwen Tu, Kumar S. Bishnupuri, Mei Han, Anguo Li, Rodney D. Newberry, Keely G. McDonald, Dan L. Crimmins, Courtney Houchen, Shrikant Anant, Brian K. Dieckgraefe

Research output: Contribution to journalArticle

18 Scopus citations

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 MutS 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.

Original languageEnglish
Pages (from-to)94-103
Number of pages10
JournalProtein Expression and Purification
Volume44
Issue number2
DOIs
StatePublished - Dec 1 2005

Keywords

  • GM-CSF
  • Hematopoietic
  • PEGylation
  • Pichia pastoris
  • Recombinant

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