The Journal of Membrane Biology ( IF 2.3 ) Pub Date : 2021-01-19 , DOI: 10.1007/s00232-020-00161-y S Jimmy Budiardjo 1 , Ayotunde Paul Ikujuni 2 , Emre Firlar 3 , Andrés Cordova 2 , Jason T Kaelber 3 , Joanna S G Slusky 1, 2
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Abstract
Overexpression of tripartite efflux pump systems in gram-negative bacteria is a principal component of antibiotic resistance. High-yield purification of the outer membrane component of these systems will enable biochemical and structural interrogation of their mechanisms of action and allow testing of compounds that target them. However, preparation of these proteins is typically hampered by low yields, requiring laborious large-scale efforts. If refolding conditions can be found, refolding these proteins from inclusion bodies can lead to increased yields as compared to membrane isolations. A classical method for refolding outer membrane proteins involves unfolding inclusion bodies in urea followed by refolding in lipid or detergent micelles. However, that method has not yet been successful in refolding tripartite efflux pump TolC. Here, we find that refolding TolC from inclusion bodies requires an additional oligomerization enhancing step of sample concentration. We show that by our method of refolding, homotrimeric TolC remains folded in SDS-PAGE, retains binding to an endogenous ligand, and recapitulates the known crystal structure by single particle cryoEM analysis. We find that TolC refolding is concentration dependent. We then extended our method to refolding CmeC, a homologous protein from Campylobacter jejuni, and find that concentration-dependent oligomerization is a general feature of these systems. Because outer membrane efflux pump components are ubiquitous across gram-negative species, we anticipate that incorporating a concentration step in refolding protocols will promote correct refolding allowing for reliable, high-yield preparation of this family of proteins.
Graphic abstract
中文翻译：
体外重折叠高产率制备外膜蛋白外排泵具有浓度依赖性
摘要
革兰氏阴性菌中三方外排泵系统的过度表达是抗生素耐药性的主要组成部分。这些系统的外膜成分的高产率纯化将能够对其作用机制进行生化和结构研究，并允许测试针对它们的化合物。然而，这些蛋白质的制备通常受到低产量的阻碍，需要进行大规模的艰苦努力。如果可以找到重折叠条件，与膜分离相比，从包涵体中重折叠这些蛋白质可以提高产量。重折叠外膜蛋白的经典方法包括在尿素中展开包涵体，然后在脂质或洗涤剂胶束中重折叠。然而，该方法在重新折叠三方外排泵 TolC 方面尚未成功。这里，我们发现从包涵体中重新折叠 TolC 需要额外的低聚增强样品浓度步骤。我们表明，通过我们的重折叠方法，同源三聚体 TolC 在 SDS-PAGE 中保持折叠，保持与内源性配体的结合，并通过单粒子 cryoEM 分析概括了已知的晶体结构。我们发现 TolC 重折叠是浓度依赖性的。然后，我们将我们的方法扩展到重新折叠 CmeC，一种来自空肠弯曲杆菌，并发现浓度依赖性寡聚化是这些系统的一般特征。由于外膜外排泵组件在革兰氏阴性菌中普遍存在，我们预计在重折叠方案中加入浓缩步骤将促进正确重折叠，从而可靠、高产地制备该蛋白质家族。
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