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Expression and purification of recombinant poly(A)-specific ribonuclease (PARN)
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Biology.
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Biology.
2006 (English)In: International Journal of Biological Macromolecules, ISSN 0141-8130, E-ISSN 1879-0003, Vol. 39, no 1-3, 95-99 p.Article in journal (Refereed) Published
Abstract [en]

PARN is a poly(A)-specific ribonuclease that degrades the poly(A) tail of mRNA. We have established conditions for expressing soluble recombinant human PARN. We investigated different Escherichia coli strains, expression vectors, media and growth conditions. We found that PARN expressed from pET33 in BL21(DE3) grown in TB and induced at OD595 approximately 1 with 1 mM IPTG yielded mg amounts of soluble PARN per litre culture. Further, a purification protocol was established to purify PARN. We use His-tag affinity chromatography, HiTrap Q HP ion exchange chromatography and 7-Me-GTP-Sepharose affinity chromatography. This purification procedure render a 90-95% pure PARN. Purified recombinant PARN has enzymatic activity and will be used for further mechanistic and structural studies.

Place, publisher, year, edition, pages
2006. Vol. 39, no 1-3, 95-99 p.
Keyword [en]
Expression of recombinant protein, His-tag purification, Ion exchange chromatography, Affinity purification by 7-Me-GTP-Sepharose, Poly(A)-specific, ribonuclease
National Category
Biological Sciences
Identifiers
URN: urn:nbn:se:uu:diva-106237DOI: 10.1016/j.ijbiomac.2006.02.025PubMedID: 16620953OAI: oai:DiVA.org:uu-106237DiVA: diva2:224295
Available from: 2009-06-17 Created: 2009-06-17 Last updated: 2017-12-13Bibliographically approved

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