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Title: Production and characterisation of human HTRA, presenilin and amyloid precursor protein in E. coli
Author: Grau, Sandra
ISNI:       0000 0004 2750 6458
Awarding Body: Cardiff University
Current Institution: Cardiff University
Date of Award: 2004
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The human serine protease L56 is thought to be a mediator in diseases such as arthritis and cancer although its precise function is unknown. L56 may also play a role in Alzheimer's disease due to an interaction with the y-secretase component presenilin 1 (PS1) which is thought to cleave C99 (a fragment of the amyloid precursor protein) to produce AP peptides. However, information regarding the function of L56 and its interaction with C99 and PS1 is limited. The present report describes the production and characterisation of recombinant L56, PS1 and C99 and their potential involvement in human disease was also investigated. Full-length 156 and a truncated form (Amac25l56) were cloned and expressed in E. coli. Amac25L56 was purified by affinity and ion exchange chromatography. Subsequent gel filtration chromatography suggested a trimeric oligomerisation state. For initial biochemical characterisation, protease assays were performed using resorufin- labelled casein as a substrate. Amac25L56 showed a typical biphasic temperature- dependent curve with an optimum of proteolytic activity at 47 C. Proteolytic activity was detected between pH 7.5 and 10.5. Furthermore, proteolytic activity increased with salt concentrations from 0 to 60 mM and remained unchanged up to 1 M NaCl. The ability of L56 to act as both a protease and chaperone was investigated in refolding assays using chemically denatured oc-amylase MalS from E. coli as a substrate. Chaperone activity could be detected for a proteolytically inactive L56 mutant (the active site serine residue was exchanged to alanine) but not for the wild type protein. Western blot analysis indicated that full-length L56 is present in synovial fluids from rheumatoid arthritis and osteoarthritis patients and in white blood cells and serum from healthy human donors. Protease assays with synovial fluid and purified Amac25L56 demonstrated the presence of at least two substrates for L56. In addition to these findings, L56 was also shown to have a potential role in Alzheimer's disease through its interaction with C99 and PS1, both of which are thought to be pivotal in the progression of this degenerative disease. Both C99 and PS1 were produced in E. coli and their interaction was verified by a stabilisation assay performed in E. coli and by co-purification using affinity chromatography. Interaction of PS1 and Amac25L56 was also confirmed by co- purification. Protease assays with purified Amac25L56, purified or membrane bound C99 and solubilised PS1 suggested that C99 is degraded by Amac25L56. Degradation was not observed in the absence of PS1, suggesting that when C99 is bound to PS1, it is no longer accessible to L56 dependent processing. These data indicate that recombinant techniques can be used to produce proteolytically active L56 and to identify interaction partners and substrates. Thus, E. coli may serve as an alternative model system to advance our understanding of the function of human proteins involved in for example Alzheimer's disease and arthritis.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available