(47e) Engineered Hepatocyte Growth Factor Mutants: New Tools for Tissue Regeneration and Vascularization | AIChE

(47e) Engineered Hepatocyte Growth Factor Mutants: New Tools for Tissue Regeneration and Vascularization

Authors 

Jones, D. S., Stanford University
Tsai, P. C., Stanford University
Cochran, J. R., Stanford University


Hepatocyte growth factor (HGF), through activation of its tyrosine kinase receptor c-MET, has shown great promise in regenerative medicine; however, the instability and poor expression yield of recombinant HGF have been a severe bottleneck to its clinical translation. Using rational and combinatorial methods, we engineered a protein fragment composed of the N terminal domain and first Kringle domain of HGF, termed NK1. Compared to full-length HGF (~105 kDa), the engineered NK1 fragment (termed eNK1) is much smaller (~20 kDa), has increased thermal stability by more than 15°C, and can be recombinantly expressed at an order of magnitude higher yield in yeast than native NK1. Dimerization of HGF’s NK1 domains is a prerequisite to c-MET dimerization and activation. Thus, to develop a more potent agonist, we created a dimeric eNK1 protein via covalent crosslinking through an N-terminal disulfide bond. Biophysical and biological characterization of the dimeric eNK1 protein will be discussed, including affinity to c-MET, downstream phosphorylation, endothelial migration, and vascularization. Remarkably, we found that the eNK1 dimers elicited significantly greater agonistic activity than the eNK1 monomer, approaching activity levels of full-length HGF. Our ultimate goal is to develop these engineered proteins as novel therapeutics for promoting tissue regeneration. Funded by NIH NCI R01CA151706.