Expression, purification and mutagenesis of recombinant class 1 Aldehyde dehydrogenase : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Molecular Biology at Massey University /
Loading...
Date
1997
DOI
Open Access Location
Authors
Journal Title
Journal ISSN
Volume Title
Publisher
Massey University
Rights
The Author
Abstract
Aldehyde dehydrogenase (AIDH) catalyses the conversion of aldehydes, for example acetaldehyde and retinal, to carboxylic acids in an NAD+- dependent reaction involved in the detoxification of aldehydes and alcohols. There are several isoenzymes, class 1 being the cytosolic form. Three over-expression and purification systems have been tested, in order to gain a high yield of active, pure class 1 aldehyde dehydrogenase. The traditional method, using T7 polymerase-driven expression in E. coli followed by ion-exchange and p-hydroxyacetophenone-affinity chromatography, gave 3 mg/L human aldehyde dehydrogenase with a high specific activity of 1.2 units/mg. Human Class 1 AIDH has been over-expressed and purified using the GST Gene Fusion System (Pharmacia Biotech), avoiding the need for AIDH-affinity chromatography and therefore allowing straight-forward purification of mutated enzymes. The GST fusion system produced 2.6 mg/L pure AIDH with a specific activity of 0.39 units/mg. The methylotrophic yeast Pichia pastoris was chosen for its high yields, in the region of grams/litre. Preparatory work was carried out with the construction of the expression plasmids and screening of the Pichia transformants. A highly conserved lysine residue (Lys-272) may be involved in acid-base catalysis of aldehyde oxidation, as well as of the esterase reaction also catalysed by AIDH. Preliminary work has been carried out on the generation of the K272A, K272R, K272H and K272L altered enzymes. The resultant activity level, kinetic behaviour and active site structure of these modified enzymes should help to elucidate further the mechanism of action of AIDH.
Description
Keywords
Aldehyde dehydrogenase, Purification, Mutagenesis