PEGylated murine Granulocyte-macrophage colony-stimulating factor: production, purification, and characterization.
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.
Sainathan
Satheesh K
SK
Division of Gastroenterology, Department of Medicine, Washington University School of Medicine, Saint Louis, MO 63110, USA.
Tu
Liwen
L
Bishnupuri
Kumar S
KS
Han
Mei
M
Li
Anguo
A
Newberry
Rodney D
RD
McDonald
Keely G
KG
Crimmins
Dan L
DL
Houchen
Courtney
C
Anant
Shrikant
S
Dieckgraefe
Brian K
BK
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
D011422:Propionates; D021382:Protein Sorting Signals; D011994:Recombinant Proteins; C021749:N-succinimidyl propionate; D011092:Polyethylene Glycols; D016178:Granulocyte-Macrophage Colony-Stimulating Factor