Methylotrophic yeast Pichia pastoris as a host for production of ATP-diphosphohydrolase (apyrase) from potato tubers (Solanum tuberosum).
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 1mg/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 48kDa was detected. The recombinant apyrase was found to function well when it was used in combination with the Pyrosequencing technology.
Nourizad
Nader
N
Department of Biotechnology, AlbaNova University center, SCFAB, Royal Institute of Technology, Roslagstullsbacken 21, SE-106 91 Stockholm, Sweden. nader@biochem.kth.se
Ehn
Maria
M
Gharizadeh
Baback
B
Hober
Sophia
S
Nyrén
Pål
P
D000429:Alcohol Oxidoreductases / Q000235:genetics; D001081:Apyrase / Q000737:chemistry* / Q000378:metabolism; D001483:Base Sequence; D015153:Blotting, Western; D002412:Cations; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D005822:Genetic Vectors; D006031:Glycosylation; D000432:Methanol / Q000494:pharmacology; D008956:Models, Chemical; D008957:Models, Genetic; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009841:Oligonucleotides / Q000235:genetics; D010710:Phosphates / Q000737:chemistry; D010843:Pichia / Q000378:metabolism*; D010957:Plasmids / Q000378:metabolism; D011401:Promoter Regions, Genetic; D011994:Recombinant Proteins / Q000737:chemistry; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D017422:Sequence Analysis, DNA; D032322:Solanum / Q000201:enzymology*
D002412:Cations; D009841:Oligonucleotides; D010710:Phosphates; D011994:Recombinant Proteins; D000429:Alcohol Oxidoreductases; C019643:alcohol oxidase; D001081:Apyrase; D000432:Methanol