Using Vibrio natriegens for high-yield production of challenging expression targets and for protein deuteration.
Using Vibrio natriegens for high-yield production of challenging expression targets and for protein deuteration.
复制标题
使用纳特里根弧菌高产生产具有挑战性的表达靶标并进行蛋白质氘化。
DOI：
10.1101/2023.11.03.565449
复制
发表时间：
2023
期刊：
影响因子：
--
通讯作者：
Krengel,Ute
中科院分区：
文献类型：
--
作者：
Mojica,Natalia;Kersten,Flore;Montserrat-Canals,Mateu;Huhn3rd,GRobb;Tislevoll,AbeloneM;Cordara,Gabriele;Teter,Ken;Krengel,Ute
可溶性蛋白质的生产对于结构/功能研究是必不可少的;然而，这通常需要毫克量的蛋白质，这可能难以用传统的表达系统获得。近年来，革兰氏阴性菌产钠弧菌（Vibrio natriegensemerged）作为一种新型的、可替代的宿主平台，用于蛋白质的高产量生产。在这里，我们使用了一种来自V的商业菌株。钠（Vmax X2），以产生毫克级的可溶性细菌和真菌蛋白，这是我们在大肠杆菌中努力实现的。这些蛋白质包括霍乱弧菌的霍乱毒素（CT）和N-乙酰葡糖胺结合蛋白A（GbpA），弗罗姆的不耐热肠毒素（LT）。大肠杆菌和灰盖鬼伞（Coprinopsis cinerea）的真菌线虫毒素CCTX 2。CT、GbpA和LT在其天然宿主中由II型分泌系统分泌。当这三种蛋白质在Vmax中产生时，它们也被分泌并且可以从生长培养基中回收。这简化了下游纯化程序，并导致与E中的生产相比显著更高的蛋白质产率。大肠杆菌（6至26倍增加）。我们还使用具有氧化氘作为溶剂的氘代基本培养基测试了蛋白质全氘代的Vmax，并且与E中的等效方案相比，实现了产量的3倍增加。杆菌这是一个好消息，因为同位素标记是昂贵的，往往是无效的，但代表了一些结构生物学技术的必要先决条件。因此，Vmax代表了一种有前途的宿主，用于生产具有挑战性的表达靶点，并以适合结构生物学研究的量进行蛋白质全氘代。
Production of soluble proteins is essential for structure/function studies; however, this usually requires milligram amounts of protein, which can be difficult to obtain with traditional expression systems. Recently, the Gram-negative bacteriumVibrio natriegensemerged as a novel and alternative host platform for production of proteins in high yields. Here, we used a commercial strain derived fromV. natriegens(Vmax X2) to produce soluble bacterial and fungal proteins in milligram scale, which we struggled to achieve inEscherichia coli. These proteins include the cholera toxin (CT) andN-acetyl glucosamine-binding protein A (GbpA) fromVibrio cholerae, the heat-labile enterotoxin (LT) fromE. coliand the fungal nematotoxin CCTX2 fromCoprinopsis cinerea. CT, GbpA, and LT are secreted by the Type II secretion system in their natural hosts. When these three proteins were produced in Vmax, they were also secreted and could be recovered from the growth media. This simplified the downstream purification procedure and resulted in considerably higher protein yields compared to production inE. coli(6- to 26-fold increase). We also tested Vmax for protein perdeuteration using deuterated minimal media with deuterium oxide as solvent and achieved a 3-fold increase in yield compared to the equivalent protocol inE. coli. This is good news, since isotopic labeling is expensive and often ineffective but represents a necessary prerequisite for some structural biology techniques. Thus, Vmax represents a promising host for production of challenging expression targets and for protein perdeuteration in amounts suitable for structural biology studies.