Expression and purification of recombinant poly(A)-specific ribonuclease (PARN)

International Journal of Biological Macromolecules
Per Nilsson, Anders Virtanen

Abstract

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.

Citations

Oct 31, 2013·Nucleic Acids Research·Maryati MaryatiG Sebastiaan Winkler
Sep 25, 2015·Nucleic Acids Research·Xiaokan ZhangFrida E Kleiman
Feb 17, 2009·Structure·Mousheng WuHaiwei Song
Sep 6, 2007·The Journal of Biological Chemistry·Per NilssonAnders Virtanen
Sep 3, 2008·Journal of Enzyme Inhibition and Medicinal Chemistry·Nikolaos A A BalatsosConstantinos Stathopoulos
Nov 22, 2019·Frontiers in Molecular Neuroscience·Jorge BaqueroFrida E Kleiman
Nov 11, 2009·The Journal of Biological Chemistry·Niklas HenrikssonAnders Virtanen
May 29, 2009·Biochemistry·Nikolaos A A BalatsosConstantinos Stathopoulos

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