title: Affinity purification of insoluble recombinant fusion proteins containing glutathione-S-transferase.
abstract: 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/glutathione-agarose system, however, is significantly limited by the frequent synthesis of recombinant proteins in insoluble 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.
authors.affiliation: Departments of Medicine, Division of Hematology and Oncology, UAB Station, University of Alabama at Birmingham, Birmingham, AL 35294-0005, USA.
authors.forename: J
authors.initials: J
authors.lastname: Hartman
authors.forename: P
authors.initials: P
authors.lastname: Daram
authors.forename: R A
authors.initials: RA
authors.lastname: Frizzell
authors.forename: T
authors.initials: T
authors.lastname: Rado
authors.forename: D J
authors.initials: DJ
authors.lastname: Benos
authors.forename: E J
authors.initials: EJ
authors.lastname: Sorscher