We report the expression of recombinant RNASET2, the only human member of the Rh/T2/S family of acid ribonucleases, in the yeast Pichia pastoris and the baculovirus-insect cell heterologous systems. In both models, the yield of recombinant protein was comparable and ranged between 5 mg/L (for a catalytically impaired mutant version of RNASET2) and 30 mg/L for the wild-type protein. Thus, the produced protein version rather than the expression system used appears to influence protein yield after optimization of culture conditions. The recombinant protein was found to undergo heterogeneous glycosylation in both systems, particularly in P. pastoris. Most importantly, the wild-type protein purified from both systems was found to be catalytically competent. The expression of recombinant RNASET2 in both systems will allow the implementation of functional assays in vivo and in vitro to better define the antioncogenic properties of this member of the Rh/T2/S ribonuclease family.

Comparison of the baculovirus-insect cell and Pichia pastoris heterologous systems for the expression of the human tumor suppressor protein RNASET2

CAMPOMENOSI, PAOLA;CINQUETTI, RAFFAELLA;TALLARITA, ELENA;TARAMELLI, ROBERTO;ACQUATI, FRANCESCO
2011

Abstract

We report the expression of recombinant RNASET2, the only human member of the Rh/T2/S family of acid ribonucleases, in the yeast Pichia pastoris and the baculovirus-insect cell heterologous systems. In both models, the yield of recombinant protein was comparable and ranged between 5 mg/L (for a catalytically impaired mutant version of RNASET2) and 30 mg/L for the wild-type protein. Thus, the produced protein version rather than the expression system used appears to influence protein yield after optimization of culture conditions. The recombinant protein was found to undergo heterogeneous glycosylation in both systems, particularly in P. pastoris. Most importantly, the wild-type protein purified from both systems was found to be catalytically competent. The expression of recombinant RNASET2 in both systems will allow the implementation of functional assays in vivo and in vitro to better define the antioncogenic properties of this member of the Rh/T2/S ribonuclease family.
RNASET2; tumor suppressor; ribonuclease; heterologous protein expression; protein glycosylation
Campomenosi, Paola; Cinquetti, Raffaella; Tallarita, Elena; Lindqvist, C.; Raimondi, I.; Grassi, P.; Näsman, J.; Dell, A.; Haslam, S. M.; Taramelli, Roberto; Acquati, Francesco
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Utilizza questo identificativo per citare o creare un link a questo documento: http://hdl.handle.net/11383/1720117
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