Expression and purification of recombinant poly(A)-specific ribonuclease (PARN).
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.
Nilsson
Per
P
Department of Cell and Molecular Biology, Uppsala University, Box 596, SE-751 24 Uppsala, Sweden. Per.Nilsson@icm.uu.se
Virtanen
Anders
A
D002846:Chromatography, Affinity / Q000379:methods; D004926:Escherichia coli / Q000235:genetics / Q000254:growth & development; D005095:Exoribonucleases / Q000235:genetics / Q000302:isolation & purification*; D006801:Humans; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification
D005095:Exoribonucleases; D011993:Recombinant Fusion Proteins