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Title: Studies on lysyl oxidase and tyrosine rich acidic matrix protein
Author: Forbes, E. G.
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 1994
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In an attempt to remove the necessity for 6M urea during the purification of lysyl oxidase, and to allow genetic manipulation of the enzyme, work was undertaken to produce active mature lysyl oxidase using recombinant DNA technology. The cDNA sequence of the rat aorta lysyl oxidase precursor was used to design primers for amplification of a DNA fragment by the polymerase chain reaction (PCR), which was then radiolabelled and used to probe a human placental cDNA library for the human lysyl oxidase sequence. During the project, the cDNA sequence of the human placental lysyl oxidase precursor was published, and primers were then designed to amplify directly, a region of the human lysyl oxidase precursor cDNA, from the codon for Asp 169 to the termination codon. This sequence is thought to encode mature lysyl oxidase. The cDNA was obtained and sequenced. The amplified DNA was cloned into a yeast shuttle vector (pDP 315), which was then used to transfect a strain of S.cerevisiae (JRY 188) and attempts were made to express mature lysyl oxidase. Only one protein band of approximately 90 kDa was observed in the yeast cell culture after running concentrated extracts on 12% SDS-PAGE. Extracts of 6M urea from porcine skin were found to contain no endogenous lysyl oxidase activity, but were able to enhance lysyl oxidase catalysed tritium release from a [4,5-3H] lysine-labelled elastin substrate. All activity was abolished in the presence of 2mM β-aminopropionitrile, a specific inhibitor of lysyl oxidase.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available