Recombinant Human GM-CSF Characterization

Introduction to Recombinant Human GM-CSF

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a type of cytokine growth factor that plays a crucial role in promoting the growth and function of white blood cells. GM-CSF specifically acts on partially committed progenitor cells to stimulate their division and differentiation along the pathways leading to the development of granulocytes and macrophages. Additionally, GM-CSF elicits a cellular response in mature end-stage cells of the granulocyte, monocyte, macrophage, and eosinophil lineages.

Fig 1. GM-CSF and monocytes/macrophages in inflammation. (Hamilton, John A. 2019)

The biological activity of GM-CSF is mediated through its interaction with cell-surface receptors. These receptors exist in both low- and high-affinity states. The low-affinity receptor, referred to as the α-subunit, is specific to GM-CSF. On the other hand, the high-affinity receptor is a heterodimer composed of both the α-subunit and a β-subunit that is shared with the receptors for other cytokines such as IL-3 and IL-5. It is important to note that the formation of the high-affinity receptor only occurs when both the α- and β-subunits are bound to GM-CSF.

Structural Studies of GM-CSF

GM-CSF is an acidic glycoprotein that belongs to the class of hematopoietic growth factors. In order to better understand the structure of non-glycosylated GM-CSF, studies were carried out using X-ray crystallography techniques. The results showed that GM-CSF consists of a bundle of four α-helices arranged in a left-handed antiparallel arrangement, and a unique folded structure, in which a double-stranded antiparallel β-sheet is fused with four α-helices, was found in GM-CSF.

Fig 2. Structure of the GM-CSF Receptor Ternary Complex. (Hansen, Guido, et al. 2008)

Protein Integrity by SDS-PAGE Silver Stain

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was utilized to assess GM-CSF protein integrity. To accomplish this, a 10%-20% linear gradient gel was used to discriminate protein species based on apparent molecular weight. Subsequently, silver staining was utilized for detection.

Fig 3. Stability evaluation by SDS-PAGE of LEUKINE®. (Pearlman, Rodney, and Y. John Wang, eds. 1996)

This method proved to be effective in identifying potential protein fragmentation and oligomerization. Notably, recombinant human GM-CSF can be divided into three major glycosylated components, bands 4, 3, and 2, which have apparent molecular weights ranging from 14 to 21 kDa. In addition, there is a highly glycosylated component, referred to as band 1, which usually exhibits a broad band above 25 kDa.

Protein Aggregation by SE-HPLC

Size exclusion high performance liquid chromatography (SE-HPLC) was utilized to determine protein aggregation in GM-CSF. The monomeric and aggregated proteins were separated based on their apparent molecular size. The separation process was performed using a Bio-Sil 125 column equipped with an isocratic aqueous solvent of 100 mM sodium phosphate and 150 mM sodium chloride at pH 7.2. To ensure accurate detection and monitoring, elution was continuously observed through absorbance readings at 220 nm. By applying this method, recombinant human GM-CSF was divided into doubled monomeric and aggregated components.

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References

1. Hamilton, John A. GM-CSF-dependent inflammatory pathways. Frontiers in immunology. 2019.10: 2055.
2. Hansen, Guido, et al. The structure of the GM-CSF receptor complex reveals a distinct mode of cytokine receptor activation. Cell. 2008. 134.3: 496-507.
3. Pearlman, Rodney, and Y. John Wang, eds. Formulation, characterization, and stability of protein drugs. Vol. 9. Springer Science & Business Media. 1996.

For research use only, not intended for any clinical use.