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Title: Expression and crystallization of the receptor-binding domains of HGF/SF
Author: Hepple, J.
Awarding Body: University of Cambridge
Current Institution: University of Cambridge
Date of Award: 1997
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Two naturally occurring partial agonists of HGF/SF exist (NK1 and NK2), which consist of the first two or three N-terminal domains of HGF/SR respectively. The aims of this thesis were: (i) to develop an expression system for the production of the NK1 and NK2 domains (ii) to characterize these recombinant domains using biochemical and biophysical techniques and assess their suitability for structural studies (iii) to crystallize these domains and collect X-ray diffraction data. Initial expression studies of the domains of HGF/SF were carried out in the prokaryotic organism, Escherichia coli. However, expression yields were limited and further reduced by extensive proteolysis. Improvements were achieved using protease-deficient strains and coexpressing bacterial folding catalysts but the final yields were insufficient for structural studies. Thus an alternative expression host, the yeast Pichia pastoris was investigated. NK1 and NK2 constructs were cloned into the expression vector cPIC-9K and integrated into the yeast's genome. Stable P. pastoris transformants containing multiple copies of the constructs were selected which expressed and secreted full length recombinant NK1 and NK2 into the supernatant. The material was purified to homogeneity using heparin affinity and ion-exchange chromatography. NK1 and NK2 were biologically active in the MDCK scatter assay. N-terminal sequencing confirmed correct processing of the signal sequence and capillary zone electrophoresis revealed NK1 and NK2 samples as 98% homogeneous. Gel filtration studies showed that NK1 existed as a monomer while NK2 consisted of monomeric and dimeric forms. Preliminary crystal trials were carried out with monomeric NK1 and NK2. NK1 crystallized under eight different conditions which were optimized to produce crystals suitable for diffraction. X-ray diffraction data sets were collected from two crystal forms under cryoconditions using rotating anode and synchrotron X-ray sources. The data sets were processed and the lattice type and unit cell dimensions for each crystal form were determined.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available