Haberl Meglič Saša, Janež Nika, Peterka Matjaž, Flisar Karel, Kotnik Tadej, Miklavčič Damijan
Faculty of Electrical Engineering, University of Ljubljana, Ljubljana, Slovenia.
Centre of Excellence for Biosensors, Instrumentation and Process Control, Centre for Biotechnology, Ajdovščina, Slovenia.
Front Bioeng Biotechnol. 2020 Sep 8;8:543187. doi: 10.3389/fbioe.2020.543187. eCollection 2020.
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 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.
基于蛋白质的技术日益多样化，这就要求生物制造进一步发展，以降低生产成本并实现产量最大化。细胞内表达的重组蛋白在纯化之前必须从生产宿主中提取出来。在几项研究中，针对不同的操作模式和形式，已证明可通过电穿孔从细菌和酵母中获取蛋白质。在此，我们测试了通过电穿孔从[具体对象未给出]中提取蛋白质的各种方案。在蛋白质产量以及裂解物中杂质（如宿主细胞DNA和内毒素）含量方面，将测试的方案与已确立的超声处理和玻璃珠研磨提取方法进行了比较。当对数生长期的细菌在37°C下处理时，无论电穿孔操作模式是分批还是连续流动，蛋白质提取产量均达到最大值。我们无法消除宿主DNA和内毒素的共提取，但使用8×1毫秒、5千伏/厘米、1赫兹的脉冲可将其降至最低。优化电穿孔后的产量（最高可达86克蛋白质/千克干重）低于超声处理（最高可达144克蛋白质/千克干重）和玻璃珠研磨（最高可达280克蛋白质/千克干重）。然而，电穿孔在很大程度上避免了细胞裂解和崩解，而在细胞裂解和崩解情况下，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，这就需要进一步纯化。
