Expression, purification and characterization of the interferon-inducible, antiviral and tumour-suppressor protein, human RNase L.
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Date
2012-03
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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.
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Keywords
2-5A, antiviral function, cellular RNA degradation, interferons, recombinant RNase L
Citation
Gupta Ankush, Rath Pramod C. Expression, purification and characterization of the interferon-inducible, antiviral and tumour-suppressor protein, human RNase L. Journal of Biosciences. 2012 Mar; 37 (1): 103-113.