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A comparative summary of expression systems for the recombinant production of galactose oxidase
KTH, Skolan för bioteknologi (BIO), Glykovetenskap. KTH, Skolan för kemivetenskap (CHE), Centra, Wallenberg Wood Science Center.
KTH, Skolan för bioteknologi (BIO), Glykovetenskap. KTH, Skolan för kemivetenskap (CHE), Centra, Wallenberg Wood Science Center.
2010 (engelsk)Inngår i: Microbial Cell Factories, ISSN 1475-2859, E-ISSN 1475-2859, Vol. 9, s. 68-Artikkel i tidsskrift (Fagfellevurdert) 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 1.1.3.9) 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.

sted, utgiver, år, opplag, sider
2010. Vol. 9, s. 68-
Emneord [en]
YEAST PICHIA-PASTORIS, HETEROLOGOUS PROTEINS, ESCHERICHIA-COLI, COFACTOR, POLYSACCHARIDES, OXIDATION, SEQUENCE, CATALYSIS, SITE
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Identifikatorer
URN: urn:nbn:se:kth:diva-26632DOI: 10.1186/1475-2859-9-68ISI: 000282454100002PubMedID: 20836876Scopus ID: 2-s2.0-77957336293OAI: oai:DiVA.org:kth-26632DiVA, id: diva2:375047
Merknad
QC 20101207Tilgjengelig fra: 2010-12-07 Laget: 2010-11-26 Sist oppdatert: 2020-03-09bibliografisk kontrollert

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