Ribonucleases form a large family of enzymes involved in RNA metabolism and are endowed with a broad range of biological functions. Amongst the different RNase proteins described in the last decades, those belonging to the Rh/T2/S subfamily show the highest degree of evolutionary conservation, suggesting the occurrence of a key critical ancestral role for this protein family. We have recently defined the human RNASET2 gene as a novel member of a group of oncosupressors called “tumor antagonizing genes”, whose activity in the control of cancer growth is carried out mainly in vivo. However, to better define the molecular pathways underlying the oncosuppressive properties of this protein, further structural and functional investigations are necessary and availability of high quality recombinant RNASET2 is of paramount importance. Here, we describe a multi-step strategy that allows production of highly pure, catalytically-competent recombinant RNASET2 in both wild-type and mutant forms. The recombinant proteins that were produced with our purification strategy will be instrumental to perform a wide range of functional assays aimed at dissecting the molecular mechanisms of RNASET2-mediated tumor suppression.

New strategies for expression and purification of recombinant human RNASET2 protein in Pichia pastoris

Lualdi, M.;TARAMELLI, ROBERTO;ACQUATI, FRANCESCO
2015

Abstract

Ribonucleases form a large family of enzymes involved in RNA metabolism and are endowed with a broad range of biological functions. Amongst the different RNase proteins described in the last decades, those belonging to the Rh/T2/S subfamily show the highest degree of evolutionary conservation, suggesting the occurrence of a key critical ancestral role for this protein family. We have recently defined the human RNASET2 gene as a novel member of a group of oncosupressors called “tumor antagonizing genes”, whose activity in the control of cancer growth is carried out mainly in vivo. However, to better define the molecular pathways underlying the oncosuppressive properties of this protein, further structural and functional investigations are necessary and availability of high quality recombinant RNASET2 is of paramount importance. Here, we describe a multi-step strategy that allows production of highly pure, catalytically-competent recombinant RNASET2 in both wild-type and mutant forms. The recombinant proteins that were produced with our purification strategy will be instrumental to perform a wide range of functional assays aimed at dissecting the molecular mechanisms of RNASET2-mediated tumor suppression.
http://www.springer.com/chemistry/biotechnology/journal/12033
Deglycosylation assay; Deletion mapping; IEX chromatography; IMAC purification; Protein tagging; Ribonuclease activity; RNase expression; SEC analysis
Lualdi, M.; Pedrini, E.; Petroni, F.; Näsman, J.; Lindqvist, C.; Scaldaferri, D.; Taramelli, Roberto; Inforzato, A.; Acquati, Francesco
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Utilizza questo identificativo per citare o creare un link a questo documento: http://hdl.handle.net/11383/2009520
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