Secretory expression of glycosylated and aglycosylated mutein of onconase from Pichia pastoris using different secretion signals and their purification and characterization.
Onconase, an RNAse extracted from embryos of the Northern leopard frog (Rana pipiens), is in a confirmatory phase IIIb clinical trial for the treatment of unresectable malignant mesothelioma. Because the current purification process for onconase is cumbersome and laborious, the development of more efficient and cost-effective alternative sources is imperative. In this study, we assessed the potential of Pichia pastoris as an expression host for the large-scale production of onconase. Because of its specific N-terminal structure, active onconase with a correct N-terminus could not be secreted by an alpha-mating factor (alpha-MF)-prepro secretion signal, and an alpha-MF-pre secretion signal should be used instead. Onconase accumulated to a high concentration (about 300 and 150 mg L(-1) for glycosylated onconase and aglycosylated mutein, respectively) in high cell density fermentation, and was purified to homogeneity with high yields (56% for glycosylated onconase and 67% for aglycosylated mutein) by a simple purification process consisting of cation exchange chromatography and size exclusion chromatography. In vitro activity assays revealed that glycosylation decreased both the RNAse activity and the cytotoxic activity of onconase. The high expression level and subsequent facile purification process make P. pastoris an efficient and cost-effective host for the large-scale production of onconase.
Department of Microorganism Engineering, Beijing Institute of Biotechnology, Beijing, China.
Hong Liang
HL
Zhao
Qing
Q
He
Chong
C
Xue
Bo
B
Sun
Xue Qin
XQ
Yao
Zhi Min
ZM
Liu
D000818:Animals; D000970:Antineoplastic Agents / Q000191:economics / Q000302:isolation & purification* / Q000378:metabolism*; D002850:Chromatography, Gel / Q000379:methods; D002852:Chromatography, Ion Exchange / Q000379:methods; D010843:Pichia / Q000235:genetics / Q000378:metabolism; D021382:Protein Sorting Signals; D011894:Rana pipiens / Q000235:genetics; D012260:Ribonucleases / Q000191:economics / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*
D000970:Antineoplastic Agents; D021382:Protein Sorting Signals; D012260:Ribonucleases; C058703:ranpirnase