rhEPO (recombinant human eosinophil peroxidase): expression in Pichia pastoris and biochemical characterization.
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.
Ciaccio
Chiara
C
Department of Experimental Medicine and Biochemical Sciences, University of Rome Tor Vergata, Via Montpellier 1, I-00133 Roma, Italy. chiara.ciaccio@uniroma2.it
Gambacurta
Alessandra
A
De Sanctis
Giampiero
G
Spagnolo
Domenico
D
Sakarikou
Christina
C
Petrella
Giovanni
G
Coletta
Massimo
M
D000818:Animals; D001681:Biological Assay; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D047209:Eosinophil Peroxidase / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D015870:Gene Expression / Q000235:genetics*; D006801:Humans; D007700:Kinetics; D008970:Molecular Weight; D010843:Pichia / Q000235:genetics*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D013057:Spectrum Analysis; D013379:Substrate Specificity; D013552:Swine
D011994:Recombinant Proteins; D047209:Eosinophil Peroxidase