Site directed mutagenesis and purification of the cDNA for human class I aldehyde dehydrogenase : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science at Massey University
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Date
1998
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Massey University
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Abstract
Aldehyde dehydrogenase (ALDH) is a key enzyme of alcohol metabolism, removing acetaldehyde which is formed as a product of the alcohol dehydrogenase reaction. If acetaldehyde is not effectively removed, acetaldehyde accumulates and produces an adverse reaction to alcohol, with nausea, flushing and increased heart rate and blood pressure. ALDH is involved in the conversion of retinal to retinoic acid (RA). RA has recently been shown to bind to receptors, which then act as nuclear transcription factors and play important roles in foetal development and maintenance of the epithelial layer in the body. Interference by ethanol and perhaps by acetaldehyde with this process is probably the cause of Foetal Alcohol Syndrome. In addition ALDH is also involved in the metabolism of catecholamine neurotransmitters, plays a role in the removal of toxic substances from the body and may have a role in protection against some chemical carcinogens. Dr. Kerrie Jones had obtained moderate levels of expression of recombinant ALDH in E. coli and constructed a number of mutants chosen on the basis of chemical modification data and sequence alignment. Mutant proteins were also expressed and assayed for enzyme activity in crude extracts. The aim of this thesis was to improve purification and yield of the expressed ALDH proteins. By the use of site-directed mutagenesis I attempted to mutate the amino acid residue Lys272 to either alanine, histidine or arginine. Future comparison of the properties of the site-directed mutants with those of the wild type enzyme will help to determine the importance of the residue (which has been replaced by mutagenesis) to catalysis.
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Keywords
Aldehyde dehydrogenase, Purification, Mutagenesis