Renaturation of a single-chain immunotoxin facilitated by chaperones and protein disulfide isomerase.
B3(Fv)-PE38KDEL, a recombinant immunotoxin, forms inclusion bodies when produced in Escherichia coli. In renaturation experiments, nonspecific aggregation of non-native polypeptide chains, and the formation of incorrect disulfide linkages lead to inactive molecules. To prevent these side reactions, we added molecular chaperones and protein disulfide isomerase (PDI) to the refolding buffer. Both DnaK and GroEL/S influenced the reactivation process. GroEL alone inhibited reactivation, but in the presence of ATP, GroEL and GroES significantly increased the yield of active protein. DnaK also increased the yield of properly folded protein and the stimulating effect of DnaK was also observed using immobilized DnaK, which can be used repeatedly without significant loss of activity. PDI, which catalyzes disulfide bridging of proteins, also stimulated reactivation of the immunotoxin. Under optimum conditions, reactivation yields in the presence of PDI were about twice that obtained with nonenzymatic disulfide bond formation. Furthermore, DnaK and PDI were additive when renaturation was performed in the presence of both proteins.
Laboratory of Molecular Biology, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892.
J
J
Buchner
U
U
Brinkmann
I
I
Pastan
D000911:Antibodies, Monoclonal; D001426:Bacterial Proteins / Q000494:pharmacology*; D018835:Chaperonin 10; D018834:Chaperonin 60; D029968:Escherichia coli Proteins; D005098:Exotoxins; D018840:HSP70 Heat-Shock Proteins; D006360:Heat-Shock Proteins / Q000494:pharmacology*; D000922:Immunotoxins / Q000737:chemistry* / Q000276:immunology / Q000633:toxicity; D007535:Isomerases / Q000378:metabolism*; D011487:Protein Conformation; D019704:Protein Disulfide-Isomerases
D000911:Antibodies, Monoclonal; C077707:B3(Fv)-PE38KDEL recombinant immunotoxin; D001426:Bacterial Proteins; D018835:Chaperonin 10; D018834:Chaperonin 60; D029968:Escherichia coli Proteins; D005098:Exotoxins; D018840:HSP70 Heat-Shock Proteins; D006360:Heat-Shock Proteins; D000922:Immunotoxins; C063842:dnaK protein, E coli; D007535:Isomerases; D019704:Protein Disulfide-Isomerases