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Methylotrophic yeast Pichia pastoris as a host for production of ATP-diphosphohydrolase (apyrase) from potato tubers (Solanum tuberosum)
KTH, Superseded Departments, Biotechnology.ORCID iD: 0000-0003-0605-8417
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2003 (English)In: Protein Expression and Purification, ISSN 1046-5928, E-ISSN 1096-0279, Vol. 27, no 2, 229-237 p.Article in journal (Refereed) Published
Abstract [en]

ATP-diphosphohydrolase (apyrase) catalyzes the hydrolysis of phosphoanhydride bonds of nucleoside tri- and di-phosphates in the presence of divalent cations. This enzyme has broad substrate specificity for nucleotides, which makes it an ideal enzyme for different biotechnical applications, such as DNA sequencing and platelet-aggregation inhibition. The only commercially available apyrase is isolated from potato tubers. To avoid batch-to-batch variations in activity and quality, we decided to produce a recombinant enzyme. The methylotrophic yeast Pichia pastoris was chosen as an eukaryotic expression host. The coding sequence of potato apyrase, without the signal peptide, was cloned into the YpDC541 vector to create a fusion with the alpha-mating secretion signal of Saccharomyces cerevisiae. The gene was placed under the control of the methanol-inducible alcohol oxidase promoter. The YpDC541-apyrase construct was integrated into P. pastoris strain SMD1168. Methanol induction resulted in secretion of apyrase to a level of 1 mg/L. The biologically active recombinant apyrase was purified by hydrophobic interaction and ion exchange chromatography. According to SDS-PAGE and Western blot analysis, the purified enzyme showed to be hyperglycosylated. By enzymatic removal of N-glycans, a single band corresponding to a molecular mass of 48 kDa was detected. The recombinant apyrase was found to function well when it was used in combination with the Pyrosequencing technology.

Place, publisher, year, edition, pages
2003. Vol. 27, no 2, 229-237 p.
Keyword [en]
smooth-muscle, ecto-atpase, purification, expression, membrane, cd39, identification, inhibitors, ec-3.6.1.5, enzyme
Identifiers
URN: urn:nbn:se:kth:diva-22302ISI: 000181405600005OAI: oai:DiVA.org:kth-22302DiVA: diva2:341000
Note
QC 20100525Available from: 2010-08-10 Created: 2010-08-10 Last updated: 2017-12-12Bibliographically approved

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Hober, Sophia

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