Growing diversity of protein-based technologies dictates further development of bio manufacturing to lower the cost of production and maximize yields. Intracellularly expressed recombinant proteins must be extracted from production host prior to purification. Use of electroporation to obtain proteins from bacteria and yeasts has been demonstrated in several studies for different modes of operation and formats. Here we tested various protocols for protein extraction from Escherichia coli by means of electroporation. The tested protocols were compared to established extraction methods of ultrasonication and glass-bead milling in terms of protein yields and content of impurities such as host cell DNA and endotoxins in the lysate. Protein extraction yield was maximal when exponentially growing bacteria were treated at 37°C, regardless of the electroporation mode of operation (batch or flow). We were unable to eliminate co-extraction of host DNA and endotoxins, but with 8 × 1 ms, 5 kV/cm, 1 Hz pulses they were minimized. Yields with optimized electroporation (up to 86 g protein/kg dry weight) were inferior to those in ultrasonication (up to 144 g protein/kg dry weight) and glass-bead milling (up to 280 g protein/kg dry weight). Nevertheless, electroporation largely avoids cell lysis and disintegration with which the extract is a mix of extracted proteins with debris of the bacterial envelope and bacterial DNA, which necessitates further purification.

中文翻译：

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电穿孔法从大肠杆菌中提取蛋白质的评价和优化


基于蛋白质的技术的日益多样化要求生物制造的进一步发展，以降低生产成本并最大限度地提高产量。细胞内表达的重组蛋白必须在纯化之前从生产宿主中提取。多项研究已证明使用电穿孔从细菌和酵母中获取蛋白质的不同操作模式和形式。在这里，我们测试了通过电穿孔从大肠杆菌中提取蛋白质的各种方案。将测试方案与现有的超声波提取和玻璃珠研磨提取方法在蛋白质产量和裂解液中宿主细胞 DNA 和内毒素等杂质含量方面进行比较。无论电穿孔操作模式（批量或流动）如何，在 37°C 下处理指数生长的细菌时，蛋白质提取率最高。我们无法消除宿主 DNA 和内毒素的共提取，但通过 8 × 1 ms、5 kV/cm、1 Hz 脉冲，它们被最小化。优化电穿孔（高达 86 g 蛋白质/kg 干重）的产量低于超声处理（高达 144 g 蛋白质/kg 干重）和玻璃珠研磨（高达 280 g 蛋白质/kg 干重）的产量。然而，电穿孔很大程度上避免了细胞裂解和崩解，其中提取物是提取的蛋白质与细菌包膜碎片和细菌 DNA 的混合物，这需要进一步纯化。