Preparation of λN-GST fusion protein for affinity immobilization of RNA.
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.
Département de Biochimie, Université de Montréal, Montreal, QC, Canada.
Geneviève
G
Di Tomasso
Philipe
P
Lampron
James G
JG
Omichinski
Pascale
P
Legault
D010582:Bacteriophage lambda; D002846:Chromatography, Affinity / Q000379:methods*; D004591:Electrophoresis, Polyacrylamide Gel; D005982:Glutathione Transferase / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D011786:Quality Control; D012313:RNA / Q000737:chemistry* / Q000302:isolation & purification* / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D014764:Viral Proteins / Q000235:genetics*
D011993:Recombinant Fusion Proteins; D014764:Viral Proteins; D012313:RNA; D005982:Glutathione Transferase