Solubilization and purification of enzymatically active glutathione S-transferase (pGEX) fusion proteins - PubMed (original) (raw)
Solubilization and purification of enzymatically active glutathione S-transferase (pGEX) fusion proteins
J V Frangioni et al. Anal Biochem. 1993 Apr.
Abstract
The pGEX glutathione S-transferase (GST) fusion protein system is used extensively for high level expression and rapid purification of fusion proteins from bacterial and eukaryotic cell lysates. Unfortunately, many GST fusion proteins are partially or completely insoluble, and thus cannot be purified efficiently from a crude lysate. We have adapted a protocol, previously used to solubilize actin, for the purification of otherwise insoluble GST fusion proteins. Using a GST fusion of the nontransmembrane protein tyrosine phosphatase 1B, we demonstrate that tyrosine phosphatase enzymatic activity is maintained during the purification process. We provide methods for the purification of GST fusion proteins at analytical and preparative scales, and demonstrate that saturation of glutathione agarose is dependent on fusion protein molecular weight. Finally, we present strategies for eluting purified fusion proteins from glutathione agarose beads, for storing eluted protein, and for preparing covalently coupled affinity matrices.
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