State Key Laboratory of Bioreactor Engineering, National Engineering Center of Biotechnology, East China University of Science and Technology, 130 Meilong Road, Shanghai 200237, People's Republic of China.
Appl Microbiol Biotechnol. 2010 Feb;85(6):1759-67. doi: 10.1007/s00253-009-2232-0. Epub 2009 Sep 16.
Previous study has shown that the degradation and aggregation of recombinant human consensus interferon-alpha mutant (cIFN) were serious when cIFN was secreted to bioreactor by Pichia pastoris. In this study, we showed that this phenomenon was concomitant well with the formation of the doublets of cIFN monomers that could be seen clearly on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The doublets were a mixture of two isomers formed by cIFN with different disulfide bonds and identified that the upper cIFN in doublets contains only one disulfide bond while the lower cIFN contains intact disulfide bonds by a novel method termed protein laddering map on SDS-PAGE. In addition, the instability of cIFN with different disulfide bond forms is also analyzed through a novel in vitro conversion assay based on incubation with different concentrations of beta-mercaptoethanol. The results showed that only a wound such as cleavage of only one disulfide bond could be fatal to cIFN stability. If the disulfide bonds in cIFN monomers were broken, three kinds of aggregates would be formed easily: covalent aggregates, non-covalent aggregates, and unknown dimers. Likewise, the unfolded species also displayed reduced stability to proteolysis. These results indicate that the incomplete formation of disulfide bond in cIFN secreted to fermentation broth triggers severe degradation and aggregation of cIFN, which result in sharp decrease of bioactivity of cIFN in bioreactor.
先前的研究表明，当重组人共识干扰素-α突变体（cIFN）通过毕赤酵母分泌到生物反应器中时，其降解和聚集非常严重。在本研究中，我们表明这种现象与 cIFN 单体二聚体的形成密切相关，在十二烷基硫酸钠聚丙烯酰胺凝胶电泳（SDS-PAGE）上可以清楚地看到二聚体。这些二聚体是由具有不同二硫键的 cIFN 形成的两种异构体的混合物，并通过一种新方法（称为 SDS-PAGE 上的蛋白梯图谱）鉴定出二聚体中的上 cIFN 仅含有一个二硫键，而下 cIFN 则含有完整的二硫键。此外，还通过基于不同浓度β-巯基乙醇孵育的新型体外转化测定法分析了具有不同二硫键形式的 cIFN 的不稳定性。结果表明，只有一个二硫键的断裂等损伤对 cIFN 稳定性是致命的。如果 cIFN 单体中的二硫键被打破，很容易形成三种聚集物：共价聚集物、非共价聚集物和未知二聚体。同样，未折叠的物种也表现出对蛋白水解的稳定性降低。这些结果表明，发酵液中分泌的 cIFN 中二硫键的不完全形成会引发 cIFN 的严重降解和聚集，从而导致生物反应器中 cIFN 的生物活性急剧下降。
