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Expresso® T7 Cloning and Expression System

产品说明书

可以快速，容易的克隆和表达困难蛋白。是基于定向PCR克隆系统所研制的克隆表达系统。

优点：
- 省时，无酶的克隆体系，利用克隆载体和细胞只需数秒完成。
- 高效：›90%的重组子。
- 严谨控制表达，N端或C端带有6组氨酸标签蛋白。
- Expressioneering™ 技术，所有Expresso®产品的特征为无连接酶克隆。

完整的克隆表达系统

Expresso® T7 克隆和蛋白表达系统是研制出的一款快速，简单，有效的定向克隆和表达PCR扩增产物的系统。系统含有预处理的pETite™ N或C端组氨酸标签克隆载体，两个感受态细胞，存在于一个单独的转化瓶中。高效HI-Control™ 10G化学感受态细胞能稳定克隆，HI-Control BL21(DE3)感受态细胞提供了严谨的可控蛋白表达。因此可以避免泄露型T7启动子系统存在的表达问题。N或C端的组氨酸标签蛋白可以使你快速的使用镍柱纯化表达蛋白。系统还具有在N端编码6xHis SUMO融合标签可以改善表达蛋白的可溶性。其他表达系统为鼠李糖启动子控制的表达。

5秒定向克隆PCR扩增基因

Expresso® T7 克隆和蛋白表达系统使用Expressioneering ™技术，是一个无酶的重组克隆策略到无缝基因载体整合过程。使用带有18bp载体互补序列的引物对靶向基因进行PCR扩增。不同于其他的限制酶方法或无连接酶方法，不需要进一步纯化和酶处理PCR产物。只需要简单混合1µl不需纯化的PCR反应产物至预处理的pETite™ T7表达载体，立即转化至HI-Control™ 10G化学感受态细胞（如图1）。

Expresso saves you a day!

克隆载体构建，使研究时间节省数十小时
全新New pETite T7 载体包含:

用于高表达的T7强启动子
用于蛋白快速纯化的位于N端或C端的组氨酸蛋白融合标签。
大小只有2.2kb，易于下游操作。
专利克隆技术增加了克隆效率。

pETite vector maps

Figure 2. pETite T7 expression vectors: Small size (2.2 kb vs. 5.4 kb for pET) for easier manipulation, including targeted mutagenesis. Vectors are pre-processed for instant enzyme-free cloning of PCR products with a choice of amino-terminal or carboxyl-terminal fusion of 6xHis tag to protein of interest.

使用HI-Control 10G 化学感受态细胞获得高效率克隆
高效的HI-Control 10G 化学感受态细胞可以确保筛选得到插入正确的克隆。对于大多数基因，> 90%的克隆都是靶向基因插入方向正确的阳性克隆（如图三）。

Figure 3. High cloning efficiency with Expresso T7 System.
Pre-processed pETite C-His vector was mixed with 1 µl of unpurified PCR gene product and transformed into HI-Control 10G Chemically Competent Cells. Colony PCR was performed on 18 randomly chosen colonies; 17 of 18 contained insert of the correct size.

HI-Control BL21(DE3)细胞控制泄漏蛋白表达
HI-Control BL21(DE3) 细胞含有高水平lac抑制物，可以保证严谨控制T7
RNA聚合酶的表达。严格控制意味着更好的适应潜在毒性基因产物（图4）。

Figure 4. Expression of various proteins using the Expresso T7 System versus a pET Vector. Genes encoding a DNA polymerase, a blue fluorescent protein (BFP), or ATP synthase b subunit (membrane protein) were cloned into pET28a or pETite vectors with N-terminal or C-terminal 6xHis tags (as indicated). pET28a clones were transformed into standard BL21(DE3) cells, and pETite clones were transformed into HI-Control BL21(DE3) cells for expression. Cultures were grown in LB at 37°C to an OD600 of 0.5 to 0.7 (odd-numbered lanes) and induced for 3 hours with 1 mM IPTG (even-numbered lanes). Cells were pelleted and lysed directly in SDS-PAGE loading buffer, and 0.05 OD equivalents were analyzed by gel electrophoresis. The gel was stained with Coomassie blue.

使用6xHis标签纯化有活性的可溶性荧光蛋白
使用T7克隆和表达系统N末端或C末端的6xHis标签获得的蛋白能够快速通过商业化镍柱进行快速纯化。图5是利用T7克隆和表达系统克隆和纯化的黄色荧光蛋白。

Expression and purification of active soluble fluorescent protein.

Figure 5. Purification of a 6xHis tagged fluorescent protein. HI-Control BL21(DE3) cells harboring pETite C-His vector containing a yellow fluorescent protein (YFP) gene were grown at 37°C in LB media to an OD600 of 0.6 (lane 1), then induced with
1 mM IPTG for 4 hours (lane 2). Cells were harvested and lysed by sonication in 300 mM NaCl, 50 mM Tris-HCl pH 8.0. Cleared lysate was loaded onto an Ni-NTA Sepharose® Column. Column flow-through (lane 3, FT) and wash (lane 4, W) fractions were collected. The bound YFP, shown in the column at left, was eluted with wash buffer containing 300 mM imidazole (lanes 5-12, E1-E8). Fluorescent fractions align with the pure protein as shown on the gel.

该产品为美国专利#6,709,861.其他相关专利的应用均属于 Lucigen 公司。
6xHis tag是被Hoffmann-La Roche,公司和, Nutley, NJ Hoffmann-LaRoche 公司授权使用，并且仅用于研究。商业授权信息来源于德国QIAGEN GmbH, QIAGEN Strasse 1, D-40724 Hilden。组氨酸标签蛋白的纯化建议使用QIAGEN公司的Ni-NTA柱子。这样子可以避免得到不好的纯化结果。

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Product Description	Size	Cat. No.
Expresso® T7 Cloning and Expression System, N-His	5 rxns	49001-1
	10 rxns	49001-2
Expresso® T7 Cloning and Expression System, C-His	5 rxns	49002-1
	10 rxns	49002-2
Expresso® T7 Cloning and Expression System, N/C-His Combo, 5 of each of N-His and C-His	10 rxns	49000-1
Expresso® T7 SUMO Cloning and Expression System	5 rxns	49003-1
	10 rxns	49003-2
HI-Control™ 10G Chemically Competent Cells (SOLOs)	12 rxns (SOLOs)	60110-1
HI-Control™ BL21(DE3) Chemically Competent Cells (SOLOs)	12 rxns (SOLOs)	60435-1
SUMO Express Protease	200 U	30801-2

ORDER INFORMATION

T7克隆和表达系统含有预处理的pETite® N-His and/or pETite C-His 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。同时还包括N端或C端组氨酸标签阳性对照插入DNA和转化阳性对照PUC DNA .

T7 SUMO克隆和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 切割控制蛋白, 鉴定克隆的正反向PCR引物。

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进口试剂

Expresso® T7 Cloning and Expression System, N-His

Expresso® T7 Cloning and Expression System

Ordering

Specifications

Manuals

Resources