Preparation of λN-GST Fusion Protein for Affinity Immobilization of RNA

Abstract

Affinity purification of in vitro transcribed RNA is becoming an attractive alternative to purification using standard denaturing gel electrophoresis. Affinity purification is particularly advantageous because it can be performed in a few hours under non-denaturing conditions. However, the performance of affinity purification methods can vary tremendously depending on the RNA immobilization matrix. It was previously shown that RNA immobilization via an optimized λN-GST fusion protein bound to glutathione-Sepharose resin allows affinity purification of RNA with very high purity and yield. This Chapter outlines the experimental procedure employed to prepare the λN-GST fusion protein used for RNA immobilization in successful affinity purifications of RNA. It describes the details of protein expression and purification as well as routine quality control analyses.

Affinity purification of T7 RNA transcripts with homogeneous ends using ARiBo and CRISPR tags

“…Small-scale affinity batch purifications were performed as described earlier (Di Tomasso et al 2011, 2012b. Briefly, 17.5 nmol of λN + -Affinity purification of RNA with homogeneous ends www.rnajournal.org 1011 L + -GST fusion protein was first added to an aliquot of the transcription reaction (75-200 µL) containing 3.5 nmol of ARiBo-fused SLI, and the volume was completed to 400 µL with equilibration buffer (50 mM HEPES at pH 7.5).…”

“…The expression and purification of the λN + -L + -GST protein was performed as described earlier (Di Tomasso et al 2011, 2012b.…”

“…The pooled fractions containing the protein of interest were dialyzed overnight at 4°C in Cse3 storage buffer (50 mM HEPES at pH 8.0, 100 mM NaCl, 2 mM DTT, and 20% glycerol). The purity (≥95%) and correct mass of the protein were verified by SDS-PAGE and mass spectroscopy, respectively, whereas its RNase-free state was verified as previously described (Di Tomasso et al 2012b). …”