Disulfide bond interchange in Escherichia coli-derived recombinant human interferon-beta 1 under denaturing conditions.
Disulfide bond interchange has been pointed out as a considerable problem in preparing recombinant proteins from Escherichia coli cells. This has been reported in the system of reducing denaturation followed by a refolding process, where incorrectly folded molecules are sometimes produced. As the possibility of disulfide bond interchange may also arise in the cytoplasm of E. coli cells, the state of sulfhydryl groups of recombinant proteins obtained from a nonreducing and nondenaturing process should be examined. The state of sulfhydryl groups of E. coli-derived recombinant human interferon-beta 1, which had been purified under nonreducing and nondenaturing conditions, was examined by using the N-(7-dimethylamino-4-methylcoumarinyl)maleimide (DACM) labeling technique. Among the three cysteine residues in E. coli-derived human interferon-beta 1, the 17th cysteine was identified as being unpaired, as in the natural molecule. However, it was found that three isomers of the recombinant protein could be formed when the protein was denatured with 6 M guanidine hydrochloride. These three isomers were identified as having unpaired cysteine residues at positions 17, 31, and 141, respectively. These results indicate that disulfide bond interchange occurs in E. coli-derived recombinant human interferon-beta 1 under denaturing conditions in spite of the absence of a reducing agent.
Kimura
S
S
Basic Research Laboratories, Toray Industries, Inc., Kanagawa.
Utsumi
J
J
Yamazaki
S
S
Shimizu
H
H
D004220:Disulfides; D005456:Fluorescent Dyes; D006801:Humans; D007370:Interferon Type I / Q000378:metabolism*; D008301:Maleimides; D010449:Peptide Mapping; D011487:Protein Conformation; D011489:Protein Denaturation; D011994:Recombinant Proteins; D013050:Spectrometry, Fluorescence; D013438:Sulfhydryl Compounds / Q000032:analysis
D004220:Disulfides; D005456:Fluorescent Dyes; D007370:Interferon Type I; D008301:Maleimides; D011994:Recombinant Proteins; D013438:Sulfhydryl Compounds; C019347:N-(7-dimethylamino-4-methylcoumarinyl)maleimide