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Expresso® Rhamnose SUMO Cloning & Expression System
Expresso® Rhamnose SUMO Cloning & Expression System
货号
49013-2
规格
10 rxns
存储温度
-20º & -80º C
产品详情
获得可溶性蛋白从未如此快速和容易
- 数秒内完成无酶定向PCR克隆。
- 节省了几天时间用于制备使用载体感受态细胞，无需连接步骤。
- 严谨控制N端6 xHis标签蛋白表达，使用可切割的SUMO可溶性标签。
- 系统可以通过T7控制表达或调控鼠李糖启动子表达。
- 表达产品的特征是无酶克隆。
完整的克隆蛋白表达系统
SUMO 克隆和蛋白表达系统的设计可以快速，容易和有效的对PCR扩增的基因进行定向克隆和可溶性表达 ，用于形成包涵体的蛋白或不容易可溶性表达的蛋白。pETite® N-His SUMO和 pRham™ N-His SUMO载体使表达的靶向蛋白氨基端带有6xHis-SUMO标签。SUMO(小的泛素样修饰体) 是一个相对小的（100残基）多肽，能够提高许多大肠菌中困难表达蛋白的可溶性表达。
The Expresso SUMO Cloning and Protein Expression Systems are based on the originalExpresso T7 and Expresso Rhamnose Cloning and Protein Expression Systems, which use Expressioneering Technology™ to provide effortless high-efficiency cloning and tightly controlled protein expression. The T7 System is complete with pre-processed pETite N-His SUMO cloning vector, and two competent cell lines, supplied in single transformation vials. High efficiency HI-Control™ 10G Chemically Competent Cells enable stable cloning and HI-Control BL21(DE3) Competent Cells provide tightly controlled protein expression, thus helping you avoid protein expression problems seen with leaky T7 promoter-based expression plasmid systems. The Rhamnose System is complete with pre-processed pRham N-His SUMO cloning vector and high-efficiency E. cloni10G Competent Cells, which are used for cloning and expression, enabling higher protein expression throughput. The N-terminal 6xHis SUMO tagged recombinant proteins can be rapidly purified using standard Nickel chromatography. SUMO Express Protease is included to provide efficient cleavage of the SUMO tag, precisely at the junction between the SUMO tag and the target protein.
5秒钟定向克隆PCR扩增的基因
SUMO蛋白克隆表达系统使用表达工程技术，无酶重组克隆策略，将基因连入表达载体采用无缝连接。PCR扩增靶向基因，并在基因两侧加入18bp与载体互补的序列。不同于限制酶方法或无连接酶克隆方法，不需要进一步的对酶处理或PCR产物进行纯化。只需要简单的混合1µl未纯化的PCR反应产物和预处理好的pETite或 pRham N-His SUMO表达载体，立即转化化学感受态细胞即可完成（如图1）
SUMO 载体包括:
- 强启动子T7用于高效表达或鼠李糖启动子用于可调控的重组蛋白表达。
- 利用N末端6×组氨酸 SUMO融合标签增加蛋白可溶性表达，并方便蛋白快速纯化。
- 载体大小为2.5 kb，易于下游操作。
- 专利的CloneSmart® 技术，增加克隆效率。
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Figure 2. SUMO expression vector: Small size (2.5 kb vs. 5.4 kb for pET) for easier manipulation, including targeted mutagenesis. Expression plasmid is pre-processed for instant enzyme-free cloning of PCR products. |
使用SUMO蛋白标签使不可溶蛋白恢复可溶活性
我们使用T7或T7sumo克隆表达系统用于表达或纯化蛋白。大规模表达研究的一些结果是为了鉴定琥珀酸放线杆菌水解酶，如图3.最初挑选了48个基因用于表达和克隆进pETite T7 C-His载体。大约这些克隆中的一半产生可溶性，具有活性的水解酶蛋白，而另外一部分是不可溶性的表达。产生非可溶性蛋白基因中五个被扩增并克隆进pETite SUMO载体，然后转化进HI-Control BL21(DE3)，其中四个恢复为可溶性，并产生活性（表1）。尽管标签移除对于水解酶活性没有必要影响，但是SUMO表达蛋白酶可以有效去除标签。
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Figure 3. Large-scale cloning and expression case study: (A) PCR products from 48 putative hydrolase genes ranging from ~1 to >3 kb. (B) Uninduced (-) and IPTG-induced (+) samples of HI-Control BL21(DE3) cells with 6 different genes cloned into the pETite C-His Vector. (C) Enhanced solubility of SUMO-tagged 2201 and 2442 gene products. Total cell extract and soluble fractions are shown. (D) Removal of 6xHis-SUMO tag from purified SUMO-2201 fusion protein by SUMO protease. –prot: uncleaved SUMO-2201 fusion protein after IMAC purification; +prot: SUMO protease-treated fusion protein; C: isolated 2201 protein after removal of 6xHis-SUMO fragment and SUMO protease by subtractive IMAC. |
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Fibrobacter succinogenes |
Soluble protein yield | |
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w/o SUMO tag |
w/SUMO tag |
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1425 |
0 mg/liter |
0 mg/liter |
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1765 |
0 mg/liter |
10 mg/liter |
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1793 |
0 mg/liter |
17 mg/liter |
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1994 |
0 mg/liter |
17 mg/liter |
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2201 |
0 mg/liter |
20 mg/liter |
Table 1. Improvement of soluble protein yield with SUMO tag. Yield of soluble protein was improved significantly for 4 of 5 Fibrobacter succinogenes genes when cloned into pETite N-His SUMO and expressed in HI-Control BL21(DE3) Cells. Cultures were induced with 1mM IPTG and grown overnight at 22°C. Yields were calculated from the amount of pure protein obtained from 100 ml of cell culture after purification over a Ni-NTA column.
切割 SUMO蛋白标签
IMAC纯化 N-His-SUMO 标签蛋白后, 标签部分可以被SUMO表达蛋白酶准确移除。SUMO表达蛋白酶识别SUMO结构而不是短的识别序列，所以可以在标签与靶蛋白间进行准确切割，不会进行脱靶切割。6xHis 标签的SUMO表达蛋白酶和切割的N-His-SUMO标签能够通过IMAC被分离并释放出靶向蛋白。
注意：盒子中包含的SUMO蛋白标签是一个特殊版本的SUMO，只能使用Lucigen的SUMO表达蛋白酶才能够进行切割。SUMO表达蛋白酶不能切割野生型的底物。因此，不推荐使用SUMO表达蛋白酶切割野生型的SUMO。
Important Product Use Information
This product is the subject of U.S. Patent #6,709,861. Additional patent applications owned by Lucigen Corporation are pending.
The 6xHis tag is licensed from Hoffmann-La Roche, Inc., Nutley, NJ and/or Hoffmann-LaRoche Ltd., Basel, Switzerland and is provided only for the use in research. Information about licenses for commercial use is available from QIAGEN GmbH, QIAGEN Strasse 1, D-40724 Hilden, Germany. Purification of 6xHis tagged proteins with Ni-NTA manufactured by QIAGEN is highly recommended for best performances and to avoid poor purification results.
SUMO Express Protease is manufactured and supplied by LifeSensors, Inc.
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T7 SUMO克隆表达系统含有预处理的pETite® N-His SUMO载体DNA, 用于克隆的HI-Control™ 10G化学感受态细胞和用于蛋白表达的HI Control BL21(DE3)化学感受态细胞。也包含SUMO阳性对照插入DNA，转化阳性对照PUC DNA，SUMO表达蛋白酶，SUMO切割对照蛋白和鉴定克隆的正反向引物。
鼠李糖SUMO克隆表达系统包含预处理的pRham N-His SUMO载体DNA，单管转化用 E. cloni 10G化学感受态细胞，自诱导试剂20%鼠李糖溶液和15%葡萄糖溶液，还包括SUMO阳性对照插入DNA，转化阳性对照PUC DNA，SUMO表达蛋白酶，SUMO切割对照蛋白和鉴定克隆的正反向引物。
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