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A comparative summary of expression systems for the recombinant production of galactose oxidase

Oliver Spadiut13, Lisbeth Olsson24 and Harry Brumer13*

Author Affiliations

1 Division of Glycoscience, School of Biotechnology, Royal Institute of Technology (KTH), SE-106 91 Stockholm, Sweden

2 Industrial Biotechnology, Department of Chemical and Biological Engineering, Chalmers University of Technology, SE-412 96 Gothenburg, Sweden

3 Wallenberg Wood Science Center, Royal Institute of Technology (KTH), SE-100 44 Stockholm, Sweden

4 Wallenberg Wood Science Center, Chalmers University of Technology, SE-412 96 Gothenburg, Sweden

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Microbial Cell Factories 2010, 9:68  doi:10.1186/1475-2859-9-68

Published: 13 September 2010



The microbes Escherichia coli and Pichia pastoris are convenient prokaryotic and eukaryotic hosts, respectively, for the recombinant production of proteins at laboratory scales. A comparative study was performed to evaluate a range of constructs and process parameters for the heterologous intra- and extracellular expression of genes encoding the industrially relevant enzyme galactose 6-oxidase (EC from the fungus Fusarium graminearum. In particular, the wild-type galox gene from F. graminearum, an optimized variant for E. coli and a codon-optimized gene for P. pastoris were expressed without the native pro-sequence, but with a His-tag either at the N- or the C-terminus of the enzyme.


The intracellular expression of a codon-optimized gene with an N-terminal His10-tag in E. coli, using the pET16b+ vector and BL21DE3 cells, resulted in a volumetric productivity of 180 U·L-1·h-1. The intracellular expression of the wild-type gene from F. graminearum, using the pPIC3.5 vector and the P. pastoris strain GS115, was poor, resulting in a volumetric productivity of 120 U·L-1·h-1. Furthermore, this system did not tolerate an N-terminal His10-tag, thus rendering isolation of the enzyme from the complicated mixture difficult. The highest volumetric productivity (610 U·L-1·h-1) was achieved when the wild-type gene from F. graminearum was expressed extracellularly in the P. pastoris strain SMD1168H using the pPICZα-system. A C-terminal His6-tag did not significantly affect the production of the enzyme, thus enabling simple purification by immobilized metal ion affinity chromatography. Notably, codon-optimisation of the galox gene for expression in P. pastoris did not result in a higher product yield (g protein·L-1 culture). Effective activation of the enzyme to generate the active-site radical copper complex could be equally well achieved by addition of CuSO4 directly in the culture medium or post-harvest.


The results indicate that intracellular production in E. coli and extracellular production in P. pastoris comprise a complementary pair of systems for the production of GalOx. The prokaryotic host is favored for high-throughput screening, for example in the development of improved enzymes, while the yeast system is ideal for production scale-up for enzyme applications.