UBC Theses and Dissertations
Construction and characterization of a chimerical cytokine which binds to cellulose: fusion of a bacterial cellulose-binding domain to steel factor Doheny, James Gregory Brian
The carboxy-terminal cellulose-binding domain (CBD) from the bacterial cellulase Cex was fused to the amino-terminal extracellular domain from the murine growth factor steel factor (SLF) to create a chimeric growth factor (SLF-CBD) which binds specifically to cellulose. The biological activity of this fusion protein was tested using the murine bone marrow cell line B6SUtA. The biological activity of the fusion protein, when free in solution, was found to be similar to that of steel factor which lacked a cellulose-binding domain. Cellulose, being inert to mammalian cells, was then used as a matrix for the immobilization of the fusion protein in situ. The adsorption of the fusion protein to cellulose in situ was accompanied by an enhancement of its biological activity. The SLF-CBD fusion protein was developed as a prototype cellulose-binding growth factor. The reasons for developing a cellulose-binding-cytokine technology were threefold. Firstly, the availability of growth factors which bind to cellulose would greatly simplify the cultivation of bone marrow cells ex vivo. The adsorption of growth factors to cellulose in vitro would provide a concentrated source of these growth factors, and reduce the net amounts required for the efficient cultivation of these cells. Secondly, growth factors which can be immobilized on the outer surfaces of cellulose-coated microcarrier beads, or on the inner surfaces of cellulose-coated rollerbottles could be used for the large scale cultivation of transformed mammalian cell lines used to produce recombinant proteins. Thirdly, cellulose as an artificial extracellular matrix, could simplify the study of receptor-cytokine interactions and signal transduction pathways by eliminating the need for specially engineered stromal cells to present immobilized growth factors to target cells.
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