New Strategies for Expression and Purification of Recombinant Human RNASET2 Protein in Pichia pastoris

Marta Lualdi, Edoardo Pedrini, Francesca Petroni, Johnny Näsman, Christer Lindqvist, Debora Scaldaferri, Roberto Taramelli, Antonio Inforzato, Francesco Acquati

Research output: Contribution to journalArticlepeer-review

Abstract

Ribonucleases form a large family of enzymes involved in RNA metabolism and are endowed with a broad range of biological functions. Among 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 oncosuppressors 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.

Original languageEnglish
Pages (from-to)513-525
Number of pages13
JournalMolecular Biotechnology
Volume57
Issue number6
DOIs
Publication statusPublished - Jun 18 2015

Keywords

  • Deglycosylation assay
  • Deletion mapping
  • IEX chromatography
  • IMAC purification
  • Protein tagging
  • Ribonuclease activity
  • RNase expression
  • SEC analysis

ASJC Scopus subject areas

  • Biochemistry
  • Biotechnology
  • Molecular Biology
  • Bioengineering
  • Applied Microbiology and Biotechnology
  • Medicine(all)

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