Disulfide bond interchange in Escherichia coli-derived recombinant human interferon-beta 1 under denaturing conditions.

Abstract:

: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.

journal_name

J Biochem

journal_title

Journal of biochemistry

authors

Kimura S,Utsumi J,Yamazaki S,Shimizu H

doi

10.1093/oxfordjournals.jbchem.a122419

subject

Has Abstract

pub_date

1988-07-01 00:00:00

pages

44-7

issue

1

eissn

0021-924X

issn

1756-2651

journal_volume

104

pub_type

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