Kimura S, Utsumi J, Yamazaki S, Shimizu H
Basic Research Laboratories, Toray Industries, Inc., Kanagawa.
J Biochem. 1988 Jul;104(1):44-7. doi: 10.1093/oxfordjournals.jbchem.a122419.
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.
二硫键互换已被指出是从大肠杆菌细胞制备重组蛋白时的一个重大问题。这在还原变性后再进行复性过程的体系中已有报道，在此过程中有时会产生错误折叠的分子。由于二硫键互换的可能性也可能在大肠杆菌细胞的细胞质中出现，因此应当检测从非还原和非变性过程获得的重组蛋白的巯基状态。使用N -（7 - 二甲基氨基 - 4 - 甲基香豆素基）马来酰亚胺（DACM）标记技术检测了在非还原和非变性条件下纯化的大肠杆菌来源的重组人干扰素 - β1的巯基状态。在大肠杆菌来源的人干扰素 - β1的三个半胱氨酸残基中，第17位半胱氨酸如同天然分子一样被鉴定为未配对。然而，发现当该蛋白用6 M盐酸胍变性时可形成三种重组蛋白异构体。这三种异构体分别被鉴定为在第17、31和141位具有未配对的半胱氨酸残基。这些结果表明，尽管不存在还原剂，但在变性条件下大肠杆菌来源的重组人干扰素 - β1中仍会发生二硫键互换。
