Affinity purification of insoluble recombinant fusion proteins containing glutathione‐S‐transferase

Academic Article


  • Prokaryotic expression of polypeptides as fusion proteins with glutathione‐S‐transferase has recently been reported as a one‐step means of purifying recombinant protein. The usefulness of the glutathione‐S‐transferase/glutathioneagarose system, however, is significantly limited by the frequent synthesis of recombinant proteins in insuluble form by Escherichia coli. We have found that for 5 separate fusion proteins containing glutathione‐S‐transferase and different domains of the large cystic fibrosis transmembrane conductance regulator, all were packaged in insoluble form by E. coli. Insolubility of these products made them inaccessible to one‐step purification utilizing this scheme requires proper folding of recombinant glutathione‐S‐transferase to allow recognition on glutathione affinity agarose, we investigated the suitability of several alternative approaches for converting insoluble recombinant fusion proteins to a soluble form amenable to glutathione‐agarose affinity purification. Low‐temperature induction of fusion protein synthesis, but not incubation with anion‐exchange resins, led to improved one‐step purification of glutathione‐S‐transferase fusion proteins from E. coli cell lysate using mild, nondenaturing conditions. Solubilization in 8 mol/L urea, but not with other chaotropic agents or detergents, also allowed preparative yields of affinity‐purified fusion protein. These techniques increase the usefulness of this recombinant protein purification scheme, and should be broadly applicable to diverse polypeptides synthesized as fusions with glutathione‐S‐transferase. Copyright © 1992 John Wiley & Sons, Inc.
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    Digital Object Identifier (doi)

    Author List

  • Hartman J; Daram P; Frizzell RA; Rado T; Benos DJ; Sorscher EJ
  • Start Page

  • 828
  • End Page

  • 832
  • Volume

  • 39
  • Issue

  • 8