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StreptoTag: A novel method for the isolation of RNA-binding proteins

  • MONIKA BACHLER (a1), RENÉE SCHROEDER (a1) and UWE VON AHSEN (a2)

Abstract

We describe a fast and simple one-step affinity-purification method for the isolation of specific RNA-binding proteins. An in vitro-transcribed hybrid RNA consisting of an aptamer sequence with high binding specificity to the antibiotic streptomycin and a putative protein-binding RNA sequence is incubated with crude extract. After complex formation, the sample is applied to an affinity column containing streptomycin immobilized to Sepharose. The binding of the in vitro-assembled RNA–protein complex to streptomycin-Sepharose is mediated by the aptamer RNA and the specifically bound proteins are recovered from the affinity matrix by elution with the antibiotic. Employing two well-characterized RNA–protein interactions, we tested the performance of this new method. The spliceosomal U1A protein and the bacteriophage MS2 coat protein could be isolated via their appropriate RNA motif containing hybrid RNA from crude yeast extracts in high yield and purity after only one round of affinity purification. As the purification principle is independent of the extract source, this new affinity chromatography strategy that makes use of an in vitro-selected antibiotic-binding RNA as a tag, “StreptoTag,” should be applicable to extracts from other organisms as well. Therefore, we propose StreptoTag to be a versatile tool for the isolation of unknown RNA-binding proteins.

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Corresponding author

Reprint requests to: Uwe von Ahsen, Dr. Bohrgasse 7b, Intercell Biotechnologies, A-1030 Vienna, Austria; e-mail: Uvonahsen@Intercell.co.at.

Keywords

StreptoTag: A novel method for the isolation of RNA-binding proteins

  • MONIKA BACHLER (a1), RENÉE SCHROEDER (a1) and UWE VON AHSEN (a2)

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