化学感受器配体结合域的高效复用与纯化方法
Summary
提出了一种 dCACHE 周质配体结合域的复用过程, 该方法由包涵体化学感受器 Tlp3 和纯化, 产生毫克量的蛋白质.
Abstract
chemoreceptors 和结构研究的天然配体的鉴定, 旨在澄清配位特异性的分子基础, 可大大促进了毫克量的纯, 折叠配体结合域的生产。试图 heterologously 表达周质配体结合域的细菌 chemoreceptors 在大肠杆菌 (大肠杆菌) 往往导致他们的目标纳入纳入机构. 本文采用空肠弯曲菌 (周质) 化学感受器 Tlp3 的 dCACHE 配体结合域为例, 提出了从包涵体中提取蛋白质、复性和纯化的方法.该方法涉及的兴趣蛋白的表达与裂解他的6标签, 分离和尿素介导 solubilisation 的包涵体, 蛋白质复用尿素耗竭, 并通过亲和层析纯化,其次是标签去除和大小排除色谱。循环二色谱光谱学用于确认纯蛋白的折叠状态。已经证明, 该协议一般是有用的生产毫克量的 dCACHE 周质配体结合域的其他细菌 chemoreceptors 在可溶性和 crystallisable 的形式。
Introduction
趋化和运动已被证明在空肠弯曲菌发病机制中发挥重要作用, 通过促进细菌的殖民和入侵主机1,2,3。随着化学信号的引导, 趋化使细菌朝着最佳的生长环境迈进。这个过程包括通过一组称为 chemoreceptors 的蛋白质 (Tlps) 识别细胞内和环境化学线索。大多数 chemoreceptors 是膜嵌入蛋白与 extracytoplasmic 配体结合域 (LBD), 跨膜领域和细胞质信令域, 后者与胞浆信号蛋白相互作用, 传输讯号到鞭毛马达4,5,6,7。
十一种不同的 chemoreceptors 已在C.菌基因组4,8中确定。到目前为止, 只有其中一些 chemoreceptors 的特点和配体特异性的 Tlp19, Tlp310,11, Tlp411, Tlp712, Tlp1113是已知的。通过生产折叠和高度纯净的重组化学感受器LBDs14、, 可以极大地促进该物种中剩余 chemoreceptors 的天然配体的鉴定, 以及其他细菌中的许多 chemoreceptors.15, 16. 然而, 在大肠杆菌中 heterologously 表达周质 LBDs 细菌 chemoreceptors 的尝试往往导致将其定向到包含机构 17, 18, 19.然而, 这种现象可以方便地分离和恢复现有的蛋白质。本文以周质化学感受器 Tlp3 的 LBD 为例, 提出了一种从包涵体中提取蛋白质、复性和纯化的方法. 之所以选择此示例, 是因为 Tlp3-LBD 属于 dCACHE 家族20 , 这些传感域大量发现在双组分组氨酸激酶和 chemoreceptors 中, 原核生物20, 21,22,23。
在这种方法中, 基于 pET151/D-TOPO 向量的表达式构造被设计为将 N 终端合并为其6标记, 后跟一个烟草蚀刻病毒 (TEV) 蛋白酶解切站点, 用于随后的标记删除19。该协议描述了蛋白在大肠杆菌中的过度表达, 分离和尿素介导的 solubilisation, 以及尿素耗竭的蛋白质复用。在复性后, 用亲和层析法纯化样品, 采用选择性标记去除和粒度排斥层析。用循环二色谱光谱法确定纯化蛋白的折叠状态。这是以前开发的方法的修改版本, 用于恢复和纯化不同化学感受器、幽门螺杆菌TlpC19 的 LBD。此过程在图 1中总结, 产生 10-20 毫克的纯, 未标记的 Tlp3-LBD 每 1 L 细菌培养, 蛋白质纯度 > 90% 按 SDS 页估计。
Protocol
1. 他的6-Tlp3-LBD 在大肠杆菌中的表达 接种150毫升不育 Luria Bertani (LB) 汤, 含有50µg 毫升-1氨苄西林, BL21 密码子加 (DE3) RIPL 细胞, 用 pET151/D-TOPO 向量变换, 用于表达其6Tlp3-LBD (氨基酸残留 42-291), 并孵化它在37°c 在一个振动筛 (轨道直径, 25 毫米) 与 200 rpm 隔夜。 准备六2升锥形烧瓶, 含800毫升不育 LB 汤和50µg 毫升-1氨苄西林。接种每瓶20毫升的隔夜文?…
Representative Results
重组表达他的6-Tlp3-LBD 在大肠杆菌导致蛋白质沉积在包涵体。在步骤2.13 中计算出的细菌培养 1 L 的表达率是其6-Tlp3-LBD 在包涵体中的大约100毫克。这里描述的蛋白质隔离过程和图 1中所说明的, 是通过亲和层析、标记去除和大小排除色谱的方法, 包括包含体的 solubilisation、蛋白质的复用和纯化。, 并产生 10-20 毫克纯, 未加标签的 T…
Discussion
本文介绍了细菌化学感受器 Tlp3 周质 LBD 中包涵体的表达和复性的简便方法。纯蛋白的制备涉及在大肠杆菌中对 pET 质粒编码基因的过度表达, 包括体的纯化和 solubilisation, 变性蛋白的复性及其纯化的连续亲和性和尺寸排除色谱步骤。尿素促进变性和稀释/透析介导的复性是该议定书的关键步骤, 最优化往往需要确保适当复存放在包含体的蛋白质26。
Tlp3-LB…
Declarações
The authors have nothing to disclose.
Acknowledgements
我们感谢 Tlp3-LBD 的早期工作。Mayra. Machuca 感激 Departamento Admistrativo de Ciencia, Tecnología e Innovación COLCIENCIAS 为博士奖学金。
Materials
| Tris base | AMRESCO | 497 | |
| Sodium chloride (NaCl) | MERK MILLIPORE | 1064041000 | |
| Ampicillin | G-BIOSCIENCES | A051-B | |
| Phenylmethanesulfonyl fluoride (PMSF) | MERCK | 52332 | |
| Triton x-100 | AMRESCO | 694 | |
| Isopropyl β-D-thiogalactoside (IPTG) | ASTRAL SCIENTIFIC PTY LTD | AST0487 | |
| Urea | AMRESCO | VWRC0568 | |
| Dithiothreitol | ASTRAL SCIENTIFIC PTY LTD | C-1029 | |
| L-arginine monohydrochloride | SIGMA-ALDRICH | A5131 | |
| Reduced L-glutathione | SIGMA-ALDRICH | G4251 | |
| Oxidized L-glutathione | SIGMA-ALDRICH | G4376 | |
| Sodium phosphate dibasic (Na2HPO4) | SIGMA-ALDRICH | 7558-79-4 | |
| Sodium phosphate monobasic (NaH2PO4) | SIGMA-ALDRICH | 10049-21-5 | |
| Ethylenediaminetetraacetic acid disodium salt dehydrate (EDTA) | AMRESCO | VWRC20302.260 | |
| Imidazole | SIGMA-ALDRICH | I2399 | |
| Glycerol | ASTRAL SCIENTIFIC PTY LTD | BIOGB0232 | |
| Nickel chloride (NiCl2) | SIGMA-ALDRICH | 339350 | |
| Glycine | AMRESCO | VWRC0167 | |
| Sodium dodecyl sulfate (SDS) | SIGMA-ALDRICH | L4509 | |
| Unstained Protein Ladder, Broad Range (10-250 kDa) | NEW ENGLAND BIOLABS | P7703 | |
| Amicon Ultracel centrifugal concentrator (Millipore) | MERCK | UFC901096 | |
| 50 mL Falcon tube | FALCON | BDAA352070 | |
| Dialysis tubing | LIVINGSTONE INTERNATIONAL PTY | Dialysis | |
| Snakeskin dialysis tubing | THERMO SCIENTIFIC™ | 68100 | |
| Prepacked HiTrap Chelating HP column | GE HEALTHCARE LIFE SCIENCES | 17-0408-01 | |
| EmulsiFlex-C5 high-pressure homogeniser | AVESTIN | EmulsiFlex™ – C5 | |
| Peristaltic Pump P-1 | GE HEALTHCARE LIFE SCIENCES | 18-1110-91 | |
| Superdex 200 HiLoad 26/60 size-exclusion column | GE HEALTHCARE LIFE SCIENCES | 28989336 | |
| JASCO J-815 spectropolarimeter | JASCO | J-815 |
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Citar este artigo
Machuca, M. A., Roujeinikova, A. Method for Efficient Refolding and Purification of Chemoreceptor Ligand Binding Domain. J. Vis. Exp. (130), e57092, doi:10.3791/57092 (2017).