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3.1.13.4: poly(A)-specific ribonuclease

This is an abbreviated version!
For detailed information about poly(A)-specific ribonuclease, go to the full flat file.

Word Map on EC 3.1.13.4

Reaction

exonucleolytic cleavage of poly(A) to 5'-AMP =

Synonyms

2',3'-exoribonuclease, 3'-exoribonuclease, adenosine-specific exonuclease, AHG2, AtPARN, BTG1-binding factor 1, Caf1, CCR4, CCR4 deadenylase complex, CCR4-associated factor 1, Ccr4-Not complex, CCR4-NOT transcription complex subunit 7, Ccr4p/Pop2p/Notp complex, CNOT6L nuclease, cytoplasmic deadenylase, deadenylase, hPAN, HsPNLDC1, MMAR_3223, More, nuclease, polyadenylate-specific exoribo-, PAB1P-dependent poly(A)-nuclease, PAN, PAN2, PAN3, PARN, PARN-1, PARN-like domain-containing protein 1, PF2046, PNLDC1, poly(A) nuclease, poly(A) nuclease complex, poly(A) ribonuclease, poly(A)-selective ribonuclease, poly(A)-specific 3'-5' ribonuclease, poly(A)-specific 3'-exoribonuclease, poly(A)-specific mRNA exoribonuclease, poly(A)-specific ribonuclease, poly(A)-specific RNase, Pop2p deadenylase, RNase AS, RNase H

ECTree

     3 Hydrolases
         3.1 Acting on ester bonds
             3.1.13 Exoribonucleases producing 5′-phosphomonoesters
                3.1.13.4 poly(A)-specific ribonuclease

Purification

Purification on EC 3.1.13.4 - poly(A)-specific ribonuclease

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PURIFICATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
by metal affinity resin
-
by Ni2+ affinity chromatography and gel filtration, purity above 98%
-
Cell lysis is performed in a buffer containing 400 mM NaCl, 50 mM Tris/HCl, pH 8.0, 2 mM ethylenediaminetetraacetic acid and 2 mM dithiothreitol. The supernatant of the lysate is loaded onto a glutathione-Sepharose column and is eluted with lysis buffer containing 30 mM reduced glutathione. Glutathione S-transferase-PARN-RNA-recognition motif is incubated with protease and a final gel filtration is performed using a buffer containing 300 mM NaCl, 20 mM Tris/HCl, pH 8.0, and 2 mM DTT. PARN-RNA-recognition motif is concentrated to 8.8 mg/ml using a vivaspin concentrator, and 7-methylguanosine triphosphate is added in 6fold molar excess. SeMet-containing PARN-RNA-recognition motif purification is analogous, with the exception that the DTT concentration is elevated to 5 mM.
His-tag affinity chromatography HiTrap Q HP and 7-Me-GTP-Sepharose affinity chromatography
-
Ni2+ matrix
-
of the recombinant protein
on Ni-NTA resin and by gel filtration, purity above 98%
-
overview on conditions
PARNn purified by glutathione-Sepharose 4B, MonoQ and Superdex 200 gel filtration columns. truncated PARN including the putative cap-binding domain purified by TALON affinity resin. full length PARN purified by Ni-NTA column
partial
-
Pop2p purified on Ni2+ column and by gel filtration, to homogeneity
purification of full-length PARN and truncated forms p62 and p54
-
purity above 99%
-
recombinant enzymes
-
recombinant GST-tagged C-terminal 133 amino acids of the PARN-1 from Escherichia coli strain BL21
-
recombinant His-tagged enzyme from Escherichia coli by nickel affinity chromatography
recombinant His-tagged enzyme from Escherichia coli strain BL21(DE3) by affinity chromatography
-
recombinant His-tagged enzyme from Escherichia coli strain BL21(DE3) by metal affinity chromatography
recombinant His-tagged enzyme from Escherichia coli strain BL21(DE3) by nickel affinity chromatography and gel filtration
recombinant His-tagged full-length enzyme and His-tagged enzyme fragment from Escherichia coli strain BL21(DE3)
recombinant His-tagged wild-type and mutant enzymes from Escherichia coli strain Rosetta by nickel affinity tandem chromatography and anion exchange chromatography
recombinant wild-type and mutant E240A, D489A, and H529A CNOT6L nuclease domains from Escherichia coli
-
The wild type and mutant forms of the cap-binding domain of PARN are overexpressed in Escherichia coli BL21(DE3) cells, harvested by centrifugation and suspended in 20 mM Tris-HCl buffer containing 1 M NaCl, 30 mM imidazole, 1 mM 1,4-DL-dithiothreitol, 0.1 mg/ml lysozyme, DNaseI and protease inhibitor cocktail, and lyse with a sonicator. Cell debris and inclusion bodies are removed by centrifugation. The supernatant is loaded on a Ni2+-NTA-agarose column and the proteins are eluted with 20 mM Tris-HCl, 1 M NaCl and 200 mM imidazole.
to homogeneity
Unlabeled and [15N], [13C]-labeled PARN cap-binding domains used for NMR experiments are synthesized by the cell-free protein expression system. After reaction, proteins are isolated by Ni2+-affinity chromatography before removing of the His6-tag. Subsequent cation-exchange chromatography yields the purified cap-binding domain.