Characterization of disulfide bonds in recombinant proteins: reduced human interleukin 2 in inclusion bodies and its oxidative refolding.
Cloned cDNA of human interleukin 2 (IL-2) was expressed in Escherichia coli cells in which IL-2 formed insoluble inclusion bodies. Human IL-2 has three Cys residues, namely, Cys-58, Cys-105, and Cys-125, and native IL-2 has an intramolecular disulfide bond between Cys-58 and Cys-105. Since the formation of inclusion bodies was thought to be due to disorder in the oxidation state of these Cys residues, all intramolecular disulfide bond isomers of IL-2 were prepared by denaturation of native IL-2 to characterize the state of a disulfide bond in IL-2 in the inclusion bodies. These isomers can be separated from native IL-2, reduced IL-2, and IL-2's with intermolecular disulfide bonds by means of reversed-phase high-performance liquid chromatography. Human IL-2 produced in inclusion bodies in E. coli carrying a recombinant DNA was analyzed by HPLC and was proved to be a fully reduced form with no intra- and intermolecular disulfide bonds. Refolding of reduced IL-2 in the presence of reduced and oxidized glutathione and a low concentration of guanidine hydrochloride resulted in the formation of the biologically active IL-2 quantitatively. Further purification provided a practically pure IL-2 preparation without contamination of any disulfide bond isomers.
D000595:Amino Acid Sequence; D004247:DNA / Q000378:metabolism; D004220:Disulfides / Q000032:analysis; D004926:Escherichia coli / Q000235:genetics; D002479:Inclusion Bodies / Q000032:analysis*; D007376:Interleukin-2 / Q000235:genetics / Q000378:metabolism; D010084:Oxidation-Reduction; D010449:Peptide Mapping; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000378:metabolism
D004220:Disulfides; D007376:Interleukin-2; D011994:Recombinant Proteins; D004247:DNA