A modified method for the purification of active large enzymes using the glutathione S-transferase expression system

The glutathione S-transferase (GST) fusion protein system is widely used for high-level expression and efficient purification of recombinant proteins from bacteria. However many GST-tagged proteins are insoluble, and the existing procedures, which employ a mixture of detergents to solubilize the mol...

Full description

Saved in:
Bibliographic Details
Published in:Analytical biochemistry 2012-02, Vol.421 (2), p.805-807
Main Authors: Deceglie, Stefania, Lionetti, Claudia, Roberti, Marina, Cantatore, Palmiro, Loguercio Polosa, Paola
Format: Article
Language:English
Subjects:
bacteria
detergents
DNA-directed RNA polymerase
DNA-Directed RNA Polymerases - biosynthesis
DNA-Directed RNA Polymerases - genetics
DNA-Directed RNA Polymerases - isolation & purification
Electrophoresis, Polyacrylamide Gel
glutathione transferase
Glutathione Transferase - biosynthesis
Glutathione Transferase - genetics
Glutathione Transferase - isolation & purification
GST fusion protein
Humans
mitochondrial RNA
Mitochondrial RNA polymerase
Protein affinity purification
purification
purification methods
Recombinant Fusion Proteins - biosynthesis
Recombinant Fusion Proteins - genetics
Recombinant Fusion Proteins - isolation & purification
recombinant proteins
tagging
Citations: Items that this one cites
Items that cite this one
Online Access:Get full text
Tags: Add Tag
No Tags, Be the first to tag this record!
Description
Summary:The glutathione S-transferase (GST) fusion protein system is widely used for high-level expression and efficient purification of recombinant proteins from bacteria. However many GST-tagged proteins are insoluble, and the existing procedures, which employ a mixture of detergents to solubilize the molecules, frequently compromise their functional activity. A further limitation is that large proteins (>80kDa) are poorly isolated by the current methods and are contaminated by truncated forms. To overcome these problems, we provide here an improved method for efficient purification of active large GST-tagged enzymes such as the 180-kDa GST-fused mitochondrial RNA polymerase.
ISSN:0003-2697
1096-0309
DOI:10.1016/j.ab.2011.12.015