Functional studies and protein engineering of hepatocyte growth factor/scatter factor

• Main Author: Holmes, Oliver
• Published: University of Cambridge 2008
• Subjects: 572.6
• Online Access: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.604185

Signalling <i>via </i>the receptor Met, HGF/SF has multiple essential roles in development and the wound response of vertebrate organisms and can activate several pathways, leading to cell survival, cell proliferation, increased motility and invasion. The aim of my research was to investigate the function of individual domains of HGF/SF and to find protein fragments with good affinity for Met. Some of these fragments were then engineered based on structural information in order to produce an antagonist of HGF/SF-Met signalling. As this antagonist could be easily produced in protein form it could avoid several of the problems associated with the gene therapy approach. In initial experiments it was found that all the domains of HGF/SF have at least some affinity for the receptor, with the N, K1 and SP domains having greatest affinity. However, only K1, K2 and K4 showed agonistic activity, whereas the K3 and SP domains showed much lower activity. Based on these experiments K1 and the N+Kl (NK1) fragment were selected for protein engineering via mutagenesis. Mutations were based on previous structural data of the NK1 dimer and scanning mutagenesis experiments. One K1 mutant, K1K (S165A/R168A), was found to possess a modest antagonistic activity. Two NK1 mutants, LE (N127A) and K1D (V140A/I142A), showed good antagonistic activity, with LE being the most promising candidate for further engineering. The work presented in this thesis clarifies the role of individual HGF/SF domains in terms of Met binding and biological activity and has led to the successful development of an NK1-based receptor antagonist. Further improvement in affinity of this antagonist via random mutagenesis and/or information from an NK1-Met complex structure are required to produce an antagonist suitable for clinical trials.