蛋白质的共表达了多方面的好处<em>大肠杆菌</em
Summary
Protein co-expression is a powerful alternative to the reconstitution in vitro of protein complexes, and is of help in performing biochemical and genetic tests in vivo. Here we report on the use of protein co-expression in Escherichia coli to obtain protein complexes, and to tune the mutation frequency of cells.
Abstract
We report here that the expression of protein complexes in vivo de Escherichia coli can be more convenient than traditional reconstitution experiments in vitro. In particular, we show that the poor solubility of Escherichia coli DNA polymerase III ε subunit (featuring 3’-5’ exonuclease activity) is highly improved when the same protein is co-expressed with the α and θ subunits (featuring DNA polymerase activity and stabilizing ε, respectively). We also show that protein co-expression in E. coli can be used to efficiently test the competence of subunits from different bacterial species to associate in a functional protein complex. We indeed show that the α subunit of Deinococcus radiodurans DNA polymerase III can be co-expressed in vivo with the ε subunit of E. coli. In addition, we report on the use of protein co-expression to modulate mutation frequency in E. coli. By expressing the wild-type ε subunit under the control of the araBAD promoter (arabinose-inducible), and co-expressing the mutagenic D12A variant of the same protein, under the control of the lac promoter (inducible by isopropyl-thio-β-D-galactopyranoside, IPTG), we were able to alter the E. coli mutation frequency using appropriate concentrations of the inducers arabinose and IPTG. Finally, we discuss recent advances and future challenges of protein co-expression in E. coli.
Introduction
表达技术的引入提高了低拷贝数酶或者生化研究和工业生产的药理学活性的蛋白质（ 如胰岛素）的。因为这些技术的出现，显著的进步已经实现，以增加重组蛋白的产量和质量。此外，原核1,2和真核3过表达系统被开发，多年来，将提供到蛋白质生物技术， 即，大肠杆菌的“工作马”有用的替代品。替代的平台，以大肠杆菌 ，特别是可获得大肠杆菌导致生产的重组肽或蛋白轴承翻译后修饰的。然而，应该指出的是，E。大肠杆菌仍然代表所选择的生物体为重组蛋白的生产。这是由于多种因素，其中最相关的可认为：I）的不少表达系统（表达载体和应变的可用性）的E.大肠杆菌 1,2; ⅱ）短的产生时间，和高生物质产量，E的大肠杆菌中的各种丰富的和合成媒体; iii）所述浅显操纵或者在生物化学，并在该微生物的基因水平; ⅳ）能产生毒性蛋白4的菌株的分离; V）株具有均匀的感应在群体水平上5,6建设。另外，最近显示适合于生产在大肠杆菌中的表达系统翻译后修饰的蛋白质的大肠杆菌可以设计和构造2。
目前，蛋白质表达主要用来获得单体或同型寡聚蛋白质，其hypersynthesis可以与单个基因克隆到合适的质粒来进行。然而，关注的是最近支付给大肠杆菌的构建大肠杆菌蛋白的共表达系统，具有挑战性的生产， 在体内的杂寡聚复合物2。有趣的是，蛋白质共同表达的早期实验解决了蓝藻核酮糖-1,5-二磷酸羧化酶/加氧7,8的大，小亚基的种间组装，和HIV-1的截断和全长形式的关联逆转录9。这些开创性的研究表明，蛋白质的共表达代表了强大的替代传统的体外重组。此外，蛋白的共表达在大肠杆菌中的大肠杆菌是用于生产不同的蛋白质轴承的翻译后修饰2，得到含非天然氨基酸的蛋白质2，并增加过表达的膜蛋白2的产率。而且，蛋白的共表达的作为工具的电位以赋予大肠杆菌大肠杆菌竞争NCE蛋白质分泌正在积极调查2。
蛋白的共表达在大肠杆菌中的两种主要的策略大肠杆菌可以追求是：i）使用单个质粒来承载不同的基因的过表达; ⅱ）在单细胞中使用多个质粒共同表达靶蛋白。在第一种情况下，该标准的质粒的选择不与那些传统的单一蛋白质表达实验不同，虽然含有串联启动子/操纵元件特定的质粒用于共表达10构成。因此，这第一种方法是相当简单的。然而，应当提到的是，使用一个单一的质粒来共表达不同蛋白质面临两个主要的困难：i）的矢量随承载基因的数量，限制共表达蛋白的数量的分子质量; ⅱ）当多个基因的单个启动子的控制下克隆，极性可以增减E从启动子远端的基因的表达。单E.采用双重或多重质粒大肠杆菌细胞具有完成所选择的载体的相容性，因此，施加约束以质粒的合格组合。然而，该第二共表达策略设有含载体的分子质量的优点，并限制极性。我们最近建造设计，方便的共表达质粒11之间的基因的穿梭蛋白共表达系统。特别是，我们构建了PGOOD矢量系列，相关特征，其中是：i）复制的一个P15A原点，以提供PGOOD质粒与含有市售载体的ColE1原点（ 例如，所述的pBAD系列12）的相容性; ⅱ）四环素抗性盒; III）LAC存在派生调控元件， 即启动操作员（O 1）夫妇和的lacI </eM> Q基因。使用适当的pBAD-PGOOD夫妇，我们能够以过表达大肠杆菌的催化核心大肠杆菌 DNA聚合酶III，三种不同的亚单位组成， 即，α（5'-3'聚合酶），ε（3'-5'外切核酸酶）和θ（稳定ε）13。特别是，我们表明，共表达αεθ复杂的是严格依赖于除大肠杆菌两者的IPTG和阿拉伯糖的大肠杆菌培养液，从PGOOD和的pBAD，分别为（ 图1A）触发过表达。
在本报告中，我们说明了如何蛋白共同表达可以有效地解决与蛋白复合物亚基的溶解性差的困难。此外，我们显示了如何在体内蛋白互补试验可以进行的，我们终于在大肠杆菌中使用的蛋白质共表达来调节突变频率报告关口我。为了这个目标，我们使用适合来说明每个案例研究相关的例子PGOOD-的pBAD夫妇。
Protocol
Representative Results
Discussion
蛋白质可以是内在无序，设有区域的三级结构不局限于构25的数量有限。这些无序的蛋白质通常容易发生聚集25，以及它们的分离和表征可能代表一个困难的任务。 E.的ε亚基大肠杆菌 DNA聚合酶III具有两个不同的结构域26,27，即：i）第N-TER域，轴承的3'-5'外切酶活性，并且在主管结合θ亚基; ⅱ）在C-之三结构域，负责结合到α（聚合酶）亚基。 ε的C之三结…
Divulgations
The authors have nothing to disclose.
Acknowledgements
由Springer和爱思唯尔的许可转载的数字大大确认。
Materials
| Name of Material/ Equipment | Company | Catalog Number | Comments/Description |
| Agar | Sigma-Aldrich | A1296 | |
| Ampicillin | Sigma-Aldrich | A9518 | |
| Chloroform | Sigma-Aldrich | 288306 | |
| EDTA | Sigma-Aldrich | EDS | |
| Glycerol | Sigma-Aldrich | G5516 | |
| INT | Sigma-Aldrich | I8377 | |
| IPTG | Sigma-Aldrich | I5502 | |
| KCl | Sigma-Aldrich | P9541 | |
| L-Arabinose | Sigma-Aldrich | A3256 | |
| MgCl2 | Sigma-Aldrich | M2670 | |
| NaCl | Sigma-Aldrich | 31434 | |
| PMSF | Sigma-Aldrich | P7626 | |
| PNP-gluc | Sigma-Aldrich | N7006 | |
| pNP-TMP | Sigma-Aldrich | T0251 | |
| Tetracyclin | Sigma-Aldrich | 87128 | |
| Trizma base | Sigma-Aldrich | T1503 | |
| Tryptone | Sigma-Aldrich | 95039 | |
| Yeast extract | Fluka | 70161 | |
| Acrylamide solution 30% | BioRad | 161-0158 | For gel electrophoresis |
| Ammonium persolphate | BioRad | 161-0700 | For gel electrophoresis |
| Glycine | BioRad | 161-0718 | For gel electrophoresis |
| SDS | BioRad | 161-0302 | For gel electrophoresis |
| TEMED | BioRad | 161-0800 | For gel electrophoresis |
| Tris | BioRad | 161-0719 | For gel electrophoresis |
| Cuvettes 0.1 cm | BioRad | 1652089 | For electroporation |
| EQUIPMENT | |||
| Centrifuge 5415R | Eppendorf | ||
| Centrifuge Allegra 21R | Beckman | ||
| Chromatography apparatus GradiFrac | Pharmacia Biotech | ||
| Gene Pulser II electroporation | BioRad | ||
| Microplate Reader 550 | Biorad | ||
| MiniProtean 3 cell | BioRad | ||
| Power Supply | BioRad | ||
| Sonicator 3000 | Misonix |
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