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Immobilization of growth factors using cellulose binding domain-cytokine fusion proteins

University of British Columbia

This study describes the application and characterization of cellulose binding domain (CBD)- cytokine fusion proteins for the cultivation of cytokine dependent cells. The affinity, binding reversibility and surface diffusivity of type-U CBDs are analyzed. Binding of CBDs to crystalline cellulose is irreversible with respect to protein dilution in the solution phase. The surface diffusion rates of an exo-β-l-4-glycanase (Cex), an endo- β -l-4-glucanase (CenA), and their respective isolated CBDs, are quantified using fluorescence recovery after photobleaching analysis. Greater than 70% of bound molecules are mobile on the cellulose surface (Dsurf ~ 3 x 10 ⁻¹¹ cm² /sec). Surface diffusion rates are dependent on surface coverage density and temperature ( Ea ~ 45 kJ/mol.K). These attributes of the C B D suggest that C B D fusion proteins are not simply immobilized on the cellulose surface, but rather localized at it, so as to retain 2- dimensional mobility. Localization of cytokines to cellulose via a C B D affinity tag, provides a convenient method for adsorbing growth factors to a solid phase (cellulose) in a surface-active form. Bioactivity and long term stability of CBD-cytokine adsorption to microcrystalline cellulose under cell culture conditions is demonstrated. Cellulose-bound fusion proteins of murine stem cell factor with CBDcex (CBD-SCF), murine interleukin-3 with CBDCenA (CBD-IL3) and murine interleukin-2 with CBDcenA (CBD-IL2), stimulate the proliferation of their respective factor dependent cells. Cellulose localization of CBD-SCF results in a significant increase in the persistence of tyrosine activation of the SCF receptor. Furthermore, when cells are incubated with cellulose-bound CBD-SCF, activated receptors and CBD-SCF co-localize at the cellulose surface. In contrast to the surface diffusion of CBDC ex, CBD-SCF does not diffuse on crystalline cellulose. This is likely a result of the formation of CBD-SCF dimers (through SCF domain dimerization) at the cellulose interface.

Thesis/Dissertation

application/pdf

2009-06-19

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http://hdl.handle.net/2429/9501

Chemical and Biological Engineering

University of British Columbia

UBCV

DSpace