Enhanced expression and purification of fungal galactose oxidase in Escherichia coli and use for analysis of a saturation mutagenesis library.
Galactose oxidase (GO) displays broad primary alcohol substrate specificity and so offers potential for engineering new substrate specificity by directed evolution. Producing variant libraries of sufficient complexity ideally requires expression of functional protein in a host such as Escherichia coli. Wild-type GO is produced by the fungus Fusarium graminiarum and is expressed poorly in E. coli. We introduced silent mutations within codons 2-7 of the mature GO coding sequence to enhance GO translation and have combined these with other expression-enhancing mutations. We selected the best E. coli host strain, autoinduction medium, induction temperature, harvest time and cell lysis procedure to produce active recombinant GO. Although normally secreted by the fungus, we have expressed GO in the cytoplasm of E. coli and have used a C-terminal Streptag II sequences for single-step affinity purification. This resulted in purification of 240 mg of functional GO per litre of shake flask culture. We have generated a saturation mutagenesis library at residue Cys383, known to influence substrate binding, and have used the optimised expression conditions to purify and characterise the resulting enzymes.
Astbury Centre for Structural Molecular Biology, Faculty of Biological Sciences, University of Leeds, Leeds LS2 9JT, UK.
Sarah E
SE
Deacon
Michael J
MJ
McPherson
D001483:Base Sequence; D020134:Catalytic Domain; D004926:Escherichia coli / Q000235:genetics*; D005670:Fusarium / Q000201:enzymology*; D005692:Galactose Oxidase / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D015971:Gene Expression Regulation, Enzymologic; D008958:Models, Molecular; D008969:Molecular Sequence Data; D015394:Molecular Structure; D016296:Mutagenesis; D054852:Small Molecule Libraries
D054852:Small Molecule Libraries; D005692:Galactose Oxidase