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Compatible-Solute-Supported Periplasmic Expression of Functional Recombinant Proteins under Stress Conditions

机译:应激条件下功能重组蛋白的相容溶质支持的周质表达

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摘要

The standard method of producing recombinant proteins such as immunotoxins (rITs) in large quantities is to transform gram-negative bacteria and subsequently recover the desired protein from inclusion bodies by intensive de- and renaturing procedures. The major disadvantage of this technique is the low yield of active protein. Here we report the development of a novel strategy for the expression of functional rIT directed to the periplasmic space of Escherichia coli. rITs were recovered by freeze-thawing of pellets from shaking cultures of bacteria grown under osmotic stress (4% NaCl plus 0.5 M sorbitol) in the presence of compatible solutes. Compatible solutes, such as glycine betaine and hydroxyectoine, are low-molecular-weight osmolytes that occur naturally in halophilic bacteria and are known to protect proteins at high salt concentrations. Adding 10 mM glycine betaine for the cultivation of E. coli under osmotic stress not only allowed the bacteria to grow under these otherwise inhibitory conditions but also produced a periplasmic microenvironment for the generation of high concentrations of correctly folded rITs. Protein purified by combinations of metal ion affinity and size exclusion chromatography was substantially stabilized in the presence of 1 M hydroxyecotine after several rounds of freeze-thawing, even at very low protein concentrations. The binding properties and cytotoxic potency of the rITs were confirmed by competitive experiments. This novel compatible-solute-guided expression and purification strategy might also be applicable for high-yield periplasmic production of recombinant proteins in different expression systems.
机译:大量生产重组蛋白(例如免疫毒素(rITs))的标准方法是转化革兰氏阴性细菌,然后通过强化的脱色和复性程序从包涵体中回收所需的蛋白。该技术的主要缺点是活性蛋白的产量低。在这里,我们报告了针对大肠杆菌周质空间的功能性rIT表达的新型策略的发展。通过在相容性溶质的存在下,在渗透压(4%NaCl加0.5 M山梨糖醇)下摇动培养的细菌,通过冻融沉淀物沉淀来回收rITs。兼容的溶质(例如甘氨酸甜菜碱和羟基ectoine)是低分子量的渗透物,存在于嗜盐细菌中,已知在高盐浓度下可以保护蛋白质。添加10 mM甘氨酸甜菜碱在渗透胁迫下培养大肠杆菌,不仅使细菌能够在这些其他抑制条件下生长,而且还产生了周质微环境,以产生高浓度的正确折叠的rIT。经过数轮冻融后,即使在非常低的蛋白质浓度下,通过金属离子亲和力和尺寸排阻色谱法组合纯化的蛋白质在1 M羟基古柯碱存在下也基本稳定。通过竞争实验证实了rIT的结合特性和细胞毒性。这种新颖的相容性溶质指导的表达和纯化策略也可能适用于在不同表达系统中重组蛋白的高产周质生产。

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