title: New Strategies for Expression and Purification of Recombinant Human RNASET2 Protein in Pichia pastoris.
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.
authors: Department of Theoretical and Applied Sciences, University of Insubria, Via JH Dunant 3, 21100, Varese, Italy, marta.lualdi@uninsubria.it.
authors: Marta
authors: M
authors: Lualdi
authors: Edoardo
authors: E
authors: Pedrini
authors: Francesca
authors: F
authors: Petroni
authors: Johnny
authors: J
authors: Näsman
authors: Christer
authors: C
authors: Lindqvist
authors: Debora
authors: D
authors: Scaldaferri
authors: Roberto
authors: R
authors: Taramelli
authors: Antonio
authors: A
authors: Inforzato
authors: Francesco
authors: F
authors: Acquati
mesh_terms: D001483:Base Sequence; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D003001:Cloning, Molecular; D017931:DNA Primers; D006031:Glycosylation; D006801:Humans; D010843:Pichia / Q000235:genetics*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D012260:Ribonucleases / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D025521:Tumor Suppressor Proteins / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification
chemical_list: D017931:DNA Primers; D011994:Recombinant Proteins; D025521:Tumor Suppressor Proteins; D012260:Ribonucleases; C425232:RNASET2 protein, human