Rezaie F, Davami F, Mansouri K, Agha Amiri S, Fazel R, Mahdian R, Davoudi N, Enayati S, Azizi M, Khalaj V
Medical Biotechnology Department, Biotechnology Research Center, Pasteur Institute of Iran, Tehran, Iran.
Medical Biology Research Center, Kermanshah University of Medical Sciences, Kermanshah, Iran.
J Appl Microbiol. 2017 Jul;123(1):134-144. doi: 10.1111/jam.13483. Epub 2017 Jun 9.
The Escherichia coli expression system is highly effective in producing recombinant proteins. However, there are some limitations in this system, especially in obtaining correctly folded forms of some complex proteins such as Fab fragments. To improve the solubility and folding quality of Fab fragments, we have examined the effect of simultaneous application of a SUMO fusion tag, EnBase cultivation mode and a redox mutant strain in the E. coli expression system.
A bicistronic gene construct was designed to express an antivascular endothelial growth factor (VEGF) Fab fragment as a model system. The construct contained a dual SUMO fusion gene fragment to encode SUMO-tagged heavy and light chains. While the expression of the construct in batch cultures of BL21 or SHuffle transformants produced insoluble and unfolded products, the induction of the transformants in EnBase medium resulted in soluble and correctly folded Fab fragment, reaching as high as 19% of the total protein in shuffle strain. The functional assays indicated that the biological activity of the target Fab is similar to the commercial anti-VEGF, Lucentis .
This study demonstrated that the combination of SUMO fusion technology, EnBase cultivation system and recruiting a redox mutant of E. coli can efficiently enhance the solubility and productivity of recombinant Fab fragments.
The presented strategy provides not only a novel method to produce soluble and active form of an anti-VEGF Fab but also may use in the efficient production of other antibody fragments.
大肠杆菌表达系统在生产重组蛋白方面非常有效。然而，该系统存在一些局限性，特别是在获得某些复杂蛋白（如Fab片段）的正确折叠形式方面。为了提高Fab片段的溶解度和折叠质量，我们研究了在大肠杆菌表达系统中同时应用SUMO融合标签、EnBase培养模式和氧化还原突变菌株的效果。
设计了一种双顺反子基因构建体，以表达抗血管内皮生长因子（VEGF）Fab片段作为模型系统。该构建体包含一个双SUMO融合基因片段，用于编码SUMO标记的重链和轻链。虽然该构建体在BL21或SHuffle转化体的分批培养中表达产生不溶性和未折叠的产物，但在EnBase培养基中诱导转化体产生了可溶性且正确折叠的Fab片段，在shuffle菌株中占总蛋白的比例高达19%。功能测定表明，目标Fab的生物活性与商业抗VEGF药物Lucentis相似。
本研究表明，SUMO融合技术、EnBase培养系统和使用大肠杆菌氧化还原突变体的组合可以有效地提高重组Fab片段的溶解度和产量。
所提出的策略不仅提供了一种生产抗VEGF Fab可溶性和活性形式的新方法，还可用于其他抗体片段的高效生产。
