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. 2022 May 5;12(9):e4401.
doi: 10.21769/BioProtoc.4401.

Expression, Purification, and in vitro Enzyme Activity Assay of a Recombinant Aldehyde Dehydrogenase from Thermus thermophilus, Using an Escherichia coli Host

Kim Shortall  1 , Edmond Magner  1 , Tewfik Soulimane  1
Affiliations

Expression, Purification, and in vitro Enzyme Activity Assay of a Recombinant Aldehyde Dehydrogenase from Thermus thermophilus, Using an Escherichia coli Host

Kim Shortall et al. Bio Protoc. .

Abstract

Based on previous in-depth characterisation, aldehyde dehydrogenases (ALDH) are a diverse superfamily of enzymes, in terms of both structure and function, present in all kingdoms of life. They catalyse the oxidation of an aldehyde to carboxylic acid using the cofactor nicotinamide adenine dinucleotide (phosphate) (NAD(P)+), and are often not substrate-specific, but rather have a broad range of associated biological functions, including detoxification and biosynthesis. We studied the structure of ALDHTt from Thermus thermophilus, as well as performed its biochemical characterisation. This allowed for insight into its potential substrates and biological roles. In this protocol, we describe ALDHTt heterologous expression in E. coli, purification, and activity assay (based on Shortall et al., 2021 ). ALDHTt was first copurified as a contaminant during caa3-type cytochrome oxidase isolation from T. thermophilus. This recombinant production system was employed for structural and biochemical analysis of wild-type and mutants, and proved efficient, yielding approximately 15-20 mg/L ALDHTt. For purification of the thermophilic his-tagged ALDHTt, heat treatment, immobilized metal affinity chromatography (IMAC), and gel filtration chromatography were used. The enzyme activity assay was performed via UV-Vis spectrophotometry, monitoring the production of reduced nicotinamide adenine dinucleotide (NADH). Graphical abstract: Flow chart outlining the steps in ALDHTt expression and purification, highlighting the approximate time required for each step.

Keywords: Aldehyde dehydrogenase; Auto-induction media; Cell culture; Enzymatic activity; Gel filtration chromatography; Heat treatment purification; Nickel affinity chromatography; UV-vis spectrophotometry.

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Conflict of interest statement

Competing interestsThere are no conflicts of interest or competing interests.

Figures

Figure 1.
Figure 1.. Elution profile of ALDHTt via Ni-affinity chromatography.
Chromatogram displaying purification of ALDHTt via Ni-affinity chromatography using a step gradient of imidazole from 50–500 mM. E. coli host proteins are eluted from 10–50 mM imidazole in the first two peaks, while ALDHTt is eluted at 200 mM imidazole in the third peak.
Figure 2.
Figure 2.. SDS-PAGE of ALDHTt expression and purification samples.
Lane 1: PageRuler Pre-stained protein ladder (ThermoFisher Scientific), lane 2: E. coli BL21(DE3) cell lysate expressing ALDHTt, lane 3: Ni-affinity chromatography 200 mM imidazole elution, lane 4: purified ALDHTt following gel filtration chromatography, lane 5: Ni-affinity chromatography flow through (adapted from Shortall et al., 2021 ).
Figure 3.
Figure 3.. Principle for ALDHTt assaying using hexanal and NAD+ with activity monitored spectrophotometrically via the appearance of NADH.
An example of the cuvette used and results obtained are demonstrated.
None
See this image and copyright information in PMC

References

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