Expression and purification of recombinant poly(A)-specific ribonuclease (PARN).
Int J Biol Macromol
; 39(1-3): 95-9, 2006 Aug 15.
Article
em En
| MEDLINE
| ID: mdl-16620953
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.
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Base de dados:
MEDLINE
Assunto principal:
Exorribonucleases
Limite:
Humans
Idioma:
En
Ano de publicação:
2006
Tipo de documento:
Article