title: Expression, purification and characterization of the interferon-inducible, antiviral and tumour-suppressor protein, human RNase L.
abstract: The interferon (IFN)-inducible, 2',5'-oligoadenylate (2-5A)-dependent ribonuclease L (RNase L) plays key role in antiviral defense of mammalian cells. Induction by IFN and activation by double-stranded RNA lead to 2-5A cofactor synthesis, which activates RNase L by causing its dimerization. Active RNase L degrades single-stranded viral as well as cellular RNAs causing apoptosis of virus-infected cells. Earlier, we had reported that expression of recombinant human RNase L caused RNA-degradation and cell-growth inhibition in E. coli without the need for exogenous 2-5A. Expression of human RNase L in E. coli usually leads to problems of leaky expression, low yield and degradation of the recombinant protein, which demands number of chromatographic steps for its subsequent purification thereby, compromising its biochemical activity. Here, we report a convenient protocol for expression of full-length, soluble and biochemically active recombinant human RNase L as GST-RNase L fusion protein from E. coli utilizing a single-step affinity purification with an appreciable yield of the highly purified protein. Recombinant RNase L was characterized by SDS-PAGE, immunoblotting and MALDI-TOF analysis. A semi-quantitative agarose-gel-based ribonuclease assay was developed for measuring its 2-5A-dependent RNase L activity against cellular large rRNAs as substrates. The optimized expression conditions minimized degradation of the protein, making it a convenient method for purification of RNase L, which can be utilized to study effects of various agents on the RNase L activity and its protein-protein interactions.
authors.affiliation: Molecular Biology Laboratory, School of Life Sciences, Jawaharlal Nehru University, New Delhi 110 067, India.
authors.forename: Ankush
authors.initials: A
authors.lastname: Gupta
authors.forename: Pramod C
authors.initials: PC
authors.lastname: Rath
mesh_terms: D000818:Animals; D015153:Blotting, Western; D017931:DNA Primers / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D004722:Endoribonucleases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D004926:Escherichia coli; D006801:Humans; D051379:Mice; D027361:Organic Anion Transporters / Q000235:genetics / Q000378:metabolism*; D020871:RNA Stability / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
chemical_list: D017931:DNA Primers; D004722:Endoribonucleases; D027361:Organic Anion Transporters; D011993:Recombinant Fusion Proteins; C487009:SLCO6A1 protein, human; C031956:2-5A-dependent ribonuclease