Hajek Philipp, Bader Annika, Helmstetter Folknand, Henke Björn, Arnold Philipp, Beitz Eric
Department of Pharmaceutical and Medicinal Chemistry, Pharmaceutical Institute, Christian-Albrechts-University Kiel, Kiel, Germany.
Anatomical Institute, Christian-Albrechts-University Kiel, Kiel, Germany.
Front Pharmacol. 2019 Apr 10;10:375. doi: 10.3389/fphar.2019.00375. eCollection 2019.
Cell-free protein production is an attractive alternative to cell-based expression. Rapid results, small-volume reactions, irrelevance of protein toxicity, flexibility, and openness of the system are strong points in favor of the cell-free system. However, the situation lacks the cellular quality control machinery comprising e.g., the translocon for inserting membrane proteins into lipid bilayers, and chaperon-assisted protein degradation pathways. Here, we compare yield and protein quality of the lactate transporter, PfFNT, from malaria parasites when produced in yeast, or in an S30-extract-based cell-free system. Besides solubilization and correct folding, PfFNT requires oligomerization into homopentamers. We assessed PfFNT folding/oligomerization and function by transmission electron microscopy imaging, transport assays, and binding of small-molecule inhibitors. For the latter, we used chromatography of the PfFNT-inhibitor complex with dual-wavelength detection, and biolayer interferometry. Our data show, that PfFNT possesses an intrinsic capability for assuming the correct fold, oligomerization pattern, and functionality during translation. This competence depended on the detergent present in the cell-free reaction. The choice of detergent further affected purification and inhibitor binding. In conclusion, in the presence of a suitable detergent, cell-free systems are very well capable of producing high quality membrane proteins.
无细胞蛋白质合成是基于细胞的表达的一种有吸引力的替代方法。快速获得结果、小体积反应、蛋白质毒性无关性、灵活性以及系统的开放性是支持无细胞系统的优点。然而，这种情况缺乏细胞质量控制机制，例如将膜蛋白插入脂质双层的转运体，以及伴侣蛋白辅助的蛋白质降解途径。在这里，我们比较了疟原虫的乳酸转运蛋白PfFNT在酵母中或基于S30提取物的无细胞系统中产生时的产量和蛋白质质量。除了溶解和正确折叠外，PfFNT还需要寡聚形成同五聚体。我们通过透射电子显微镜成像、转运测定以及小分子抑制剂的结合来评估PfFNT的折叠/寡聚化和功能。对于后者，我们使用了具有双波长检测的PfFNT-抑制剂复合物色谱法和生物层干涉术。我们的数据表明，PfFNT在翻译过程中具有呈现正确折叠、寡聚化模式和功能的内在能力。这种能力取决于无细胞反应中存在的去污剂。去污剂的选择进一步影响了纯化和抑制剂结合。总之，在存在合适去污剂的情况下，无细胞系统非常有能力生产高质量的膜蛋白。
