Dynamic localization and persistent stimulation of factor-dependent cells by a stem cell factor / cellulose binding domain fusion protein.
The extracellular matrix provides structural components that support the development of tissue morphology and the distribution of growth factors that modulate the overall cellular response to those growth factors. The ability to manipulate the presentation of factors in culture systems should provide an additional degree of control in regulating the stimulation of factor-dependent cells for tissue engineering applications. Cellulose binding domain (CBD) fusion protein technology facilitates the binding of bioactive cytokines to cellulose materials, and has permitted the analysis of several aspects of cell stimulation by surface-localized growth factors. We previously reported the synthesis and initial characterization of a fusion protein comprised of a CBD and murine stem cell factor (SCF) (Doheny et al. [1999] Biochem J 339:429-434). A significant advantage of the CBD fusion protein system is that it permits the stimulation of factor-dependent cells with localized growth factor, essentially free of nonfactor-derived interactions between the cell and matrix. In this work, the long-term stability and bioactivity of SCF-CBD fusions adsorbed to microcrystalline cellulose under cell culture conditions is demonstrated. Cellulose-bound SCF-CBD is shown to stimulate receptor polarization in the cell membrane and adherence to the cellulose matrix. In addition, cellulose-surface presentation of the SCF-CBD attenuates c-kit dephosphorylation kinetics, potentially modulating the overall response of the cell to the SCF signal.
Jervis
Eric J
EJ
Protein Engineering Network of Centers of Excellence, Biotechnology Laboratory and the Department of Chemical and Biological Engineering, The University of British Columbia, Vancouver, BC, V6T 1Z3, Canada.
Guarna
M Marta
MM
Doheny
J Greg
JG
Haynes
Charles A
CA
Kilburn
Douglas G
DG
D000818:Animals; D018929:Cell Culture Techniques; D002462:Cell Membrane / Q000187:drug effects; D002478:Cells, Cultured; D002480:Cellulase / Q000737:chemistry* / Q000235:genetics; D002482:Cellulose / Q000737:chemistry*; D004795:Enzyme Stability; D004800:Enzymes, Immobilized / Q000737:chemistry* / Q000235:genetics; D051379:Mice; D010766:Phosphorylation / Q000187:drug effects; D017434:Protein Structure, Tertiary; D019009:Proto-Oncogene Proteins c-kit / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000494:pharmacology; D019089:Stem Cell Factor / Q000235:genetics / Q000494:pharmacology*; D013234:Stem Cells / Q000187:drug effects / Q000201:enzymology; D023822:Tissue Engineering
D004800:Enzymes, Immobilized; D011993:Recombinant Fusion Proteins; D019089:Stem Cell Factor; D002482:Cellulose; D019009:Proto-Oncogene Proteins c-kit; D002480:Cellulase; C109691:microcrystalline cellulose