New Strategies for Expression and Purification of Recombinant Human RNASET2 Protein in Pichia pastoris
| Autor: | Johnny Näsman, Christer Lindqvist, Edoardo Pedrini, Francesco Acquati, Debora Scaldaferri, Marta Lualdi, Francesca Petroni, Roberto Taramelli, Antonio Inforzato |
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| Rok vydání: | 2015 |
| Předmět: |
Glycosylation
Subfamily Protein family RNase P Mutant Bioengineering Computational biology Biology Applied Microbiology and Biotechnology Biochemistry Chromatography Affinity Pichia Conserved sequence law.invention Pichia pastoris Ribonucleases law Humans Cloning Molecular RNase expression Molecular Biology Gene DNA Primers Protein tagging Genetics Deletion mapping Base Sequence Tumor Suppressor Proteins Ribonuclease activity IMAC purification biology.organism_classification Recombinant Proteins Deglycosylation assay IEX chromatography SEC analysis Chromatography Gel Recombinant DNA Biotechnology |
| Zdroj: | Molecular Biotechnology. 57:513-525 |
| ISSN: | 1559-0305 1073-6085 |
| DOI: | 10.1007/s12033-015-9845-6 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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