A Pichia pastoris expression system has for the first time been successfully developed to produce rhEPO (recombinant human eosinophil peroxidase). The full-length rhEPO coding sequence was cloned into the pPIC9 vector in frame with the yeast α-Factor secretion signal under the transcriptional control of the AOX (acyl-CoA oxidase) promoter, and transformed into P. pastoris strain GS115. Evidence for the production of rhEPO by P. pastoris as a glycosylated dimer precursor of approx. 80 kDa was determined by SDS/PAGE and gel filtration chromatography. Recombinant hEPO undergoes proteolytic processing, similar to that in the native host, to generate two chains of approx. 50 and 20 kDa. A preliminary biochemical characterization of purified rhEPO demonstrated that the spectral and kinetic properties of the recombinant wild-type EPO are comparable with those of the native enzyme and are accompanied by oxidizing activity towards several physiological anionic substrates such as SCN−, Br− and Cl−. On the basis of the estimated Km and kcat values it is evident that the pseudohalide SCN− is the most specific substrate for rhEPO, consistent with the catalytic properties of other mammalian EPOs purified from blood.
rhEPO (recombinant human eosinophil peroxidase): expression in Pichia pastoris and biochemical characterization
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Chiara Ciaccio, Alessandra Gambacurta, Giampiero DE Sanctis, Domenico Spagnolo, Christina Sakarikou, Giovanni Petrella, Massimo Coletta; rhEPO (recombinant human eosinophil peroxidase): expression in Pichia pastoris and biochemical characterization. Biochem J 15 April 2006; 395 (2): 295–301. doi: https://doi.org/10.1042/BJ20051385
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