Change search
ReferencesLink to record
Permanent link

Direct link
A comparative summary of expression systems for the recombinant production of galactose oxidase
KTH, School of Biotechnology (BIO), Glycoscience. KTH, School of Chemical Science and Engineering (CHE), Centres, Wallenberg Wood Science Center.
KTH, School of Biotechnology (BIO), Glycoscience. KTH, School of Chemical Science and Engineering (CHE), Centres, Wallenberg Wood Science Center.
2010 (English)In: Microbial Cell Factories, ISSN 1475-2859, Vol. 9, 68- p.Article in journal (Refereed) Published
Abstract [en]

Background: 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 Results: The intracellular expression of a codon-optimized gene with an N-terminal His(10)-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 His(10)-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 pPICZ alpha-system. A C-terminal His(6)-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. Conclusions: 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.

Place, publisher, year, edition, pages
2010. Vol. 9, 68- p.
Keyword [en]
National Category
Industrial Biotechnology
URN: urn:nbn:se:kth:diva-26632DOI: 10.1186/1475-2859-9-68ISI: 000282454100002ScopusID: 2-s2.0-77957336293OAI: diva2:375047
QC 20101207Available from: 2010-12-07 Created: 2010-11-26 Last updated: 2010-12-07Bibliographically approved

Open Access in DiVA

No full text

Other links

Publisher's full textScopus

Search in DiVA

By author/editor
Spadiut, OliverBrumer, Harry
By organisation
GlycoscienceWallenberg Wood Science Center
In the same journal
Microbial Cell Factories
Industrial Biotechnology

Search outside of DiVA

GoogleGoogle Scholar
The number of downloads is the sum of all downloads of full texts. It may include eg previous versions that are now no longer available

Altmetric score

Total: 24 hits
ReferencesLink to record
Permanent link

Direct link