Engineering of Artificial Biomatrix through Extracellular Matrix Targeting of Functional Proteins

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Novel protocols for engineering of artificial biomatrix by targeting of proteins to the extracellular matrices have been developed. First, we established a protocol to target transforming growth factor-alpha (TGF-a) to the fibronectin matrix via chimerization with the marix assembly domain of fibronectin. The resulting chimeric protein retains the mitogenic activity of TGF-a without-compromising its matrix assembly activity. The chimeric protein was more potent than authentic, soluble TGF-a in promoting would healing of rabbit cornea, demonstrating its potency as a new protein drug for accelerating, wound healing. Second, we developed another protocol to target functional proteins to the basement membrane, the scaffold for growth and differentiation of stem cells. We utilized the N-terminal laminin-binding domain of agrin to target hepatocyte growth factor (HGF) or green fluorescent protein to the basement membrane. The resulting chimeric protein retained the motogenic activity of HGF … More as well as the laminin-binding activity of agrin. When cells expressing the chimeric protein were grown on the laminin-containing basement membrane-like gel (I.e., Matrigel), the chimeric protein was found to be deposited around the cells. This protocol will be applicable for targeting of various growth factors and/or cell differentiation-inducing factors to the basement membranes of vasculature and other organs. Third, we purified new laminin isoforms, I.e., laminin-8 and -10, and characterized their physiologic activities with special reference to integrin-mediated signaling events. We also established the expression system for laminin-6, -8, and -10 by cDNA transfection into human 293 cells. We also succeeded in expression of the functional domains of laminin chains with putative activities for integrin/alpha-dystroglycan binding as well as laminin selfassembly. These domains of laminin chains will be useful for construction of tailor-made, artificial basement membranes optimized for proliferation and differentiation of particular cell types, e.g., embryonic and tissue stem cells. Less