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Affinity purification of insoluble recombinant fusion proteins containing glutathione‐ S ‐transferase
Author(s) -
Hartman John,
Daram Paru,
Frizzell Raymond A.,
Rado Thomas,
Benos Dale J.,
Sorscher Eric J.
Publication year - 1992
Publication title -
biotechnology and bioengineering
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.136
H-Index - 189
eISSN - 1097-0290
pISSN - 0006-3592
DOI - 10.1002/bit.260390805
Subject(s) - glutathione , fusion protein , glutathione s transferase , biochemistry , recombinant dna , escherichia coli , affinity chromatography , chemistry , agarose , transferase , tandem affinity purification , chaotropic agent , protein purification , chromatography , enzyme , gene
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.