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EnglishA 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 alpha-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 K(m) 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. / CIACCIO C; GAMBACURTA A; G. DE SANCTIS; SPAGNOLO D; SAKARIKOU C; PETRELLA G; COLETTA M. - In: BIOCHEMICAL JOURNAL. - ISSN 0264-6021. - 395:2(2006), pp. 295-301.
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| Titolo: | rhEPO (recombinant human eosinophil peroxidase): expression in Pichia pastoris and biochemical characterization. |
| Autori: | |
| Data di pubblicazione: | 2006 |
| Rivista: | |
| Citazione: | rhEPO (recombinant human eosinophil peroxidase): expression in Pichia pastoris and biochemical characterization. / CIACCIO C; GAMBACURTA A; G. DE SANCTIS; SPAGNOLO D; SAKARIKOU C; PETRELLA G; COLETTA M. - In: BIOCHEMICAL JOURNAL. - ISSN 0264-6021. - 395:2(2006), pp. 295-301. |
| Abstract: | 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 alpha-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 K(m) 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. |
| Handle: | http://hdl.handle.net/11581/200801 |
| Appare nelle tipologie: | Articolo |
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