Matos Cristina F R O, Robinson Colin, Alanen Heli I, Prus Piotr, Uchida Yuko, Ruddock Lloyd W, Freedman Robert B, Keshavarz-Moore Eli
Centre for Molecular Processing, School of Biosciences, University of Kent, Canterbury CT2 7NJ, U.K.
Biotechnol Prog. 2014 Mar-Apr;30(2):281-90. doi: 10.1002/btpr.1858. Epub 2014 Jan 22.
Numerous high-value therapeutic proteins are produced in Escherichia coli and exported to the periplasm, as this approach simplifies downstream processing and enables disulfide bond formation. Most recombinant proteins are exported by the Sec pathway, which transports substrates across the plasma membrane in an unfolded state. The Tat system also exports proteins to the periplasm, but transports them in a folded state. This system has attracted interest because of its tendency to transport correctly folded proteins, but this trait renders it unable to export proteins containing disulfide bonds since these are normally acquired only in the periplasm; reduced substrates tend to be recognized as incorrectly folded and rejected. In this study we have used a series of novel strains (termed CyDisCo) which oxidise disulfide bonds in the cytoplasm, and we show that these cells efficiently export a range of disulfide-containing proteins when a Tat signal peptide is attached. These test proteins include alkaline phosphatase (PhoA), a phytase containing four disulfide bonds (AppA), an antiinterleukin 1β scFv and human growth hormone. No export of PhoA or AppA is observed in wild-type cells lacking the CyDisCo factors. The PhoA, AppA and scFv proteins were exported in an active form by Tat in the CyDisCo strain, and mass spectrometry showed that the vast majority of the scFv protein was disulfide-bonded and correctly processed. The evidence indicates that this combination of Tat + CyDisCo offers a novel means of exporting active, correctly folded disulfide bonded proteins to the periplasm.
许多高价值治疗性蛋白质在大肠杆菌中产生并输出到周质，因为这种方法简化了下游加工并能形成二硫键。大多数重组蛋白通过Sec途径输出，该途径以未折叠状态将底物转运过质膜。Tat系统也将蛋白质输出到周质，但以折叠状态转运它们。由于其倾向于转运正确折叠的蛋白质，该系统引起了人们的兴趣，但这一特性使其无法输出含有二硫键的蛋白质，因为这些二硫键通常仅在周质中形成；还原的底物往往被识别为错误折叠并被拒之门外。在本研究中，我们使用了一系列新型菌株（称为CyDisCo），它们在细胞质中氧化二硫键，并且我们表明，当连接Tat信号肽时，这些细胞能有效地输出一系列含二硫键的蛋白质。这些测试蛋白包括碱性磷酸酶（PhoA）、含有四个二硫键的植酸酶（AppA）、抗白细胞介素1β单链抗体片段和人生长激素。在缺乏CyDisCo因子的野生型细胞中未观察到PhoA或AppA的输出。PhoA、AppA和单链抗体片段蛋白在CyDisCo菌株中通过Tat以活性形式输出，质谱分析表明，绝大多数单链抗体片段蛋白形成了二硫键并得到了正确加工。证据表明，Tat + CyDisCo的这种组合提供了一种将活性、正确折叠的二硫键结合蛋白输出到周质的新方法。
