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Expresso® Rhamnose Cloning & Expression System, N-His

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Expresso® Rhamnose Cloning & Expression System, N-His

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产品名称:

Expresso® Rhamnose Cloning & Expression System, N-His

货号
49011-1
规格
5 rxns
存储温度
-20º & -80º C
没有此类产品
产品描述

Expresso® Rhamnose Cloning & Protein Expression System

 

产品说明书

 

利用午后时间毫不费力的克隆一个PCR扩增基因,并可在第二天表达重组蛋白。

  • 五秒,定向,无酶PCR克隆,获得90%重组子
  • 单感受态细胞宿主菌株用于克隆和表达。
  • 使用克隆的鼠李糖启动子严谨控制表达。
  • 使用自诱导试剂获得高产出。
  • Expressioneering™ Technology表达产品特征是无连接克隆。

 

使用 Expressioneering™技术的完整克隆表达系统
鼠李糖克隆和蛋白表达系统是用于快速,容易和有效的定向克隆和表达PCR扩增基因用于蛋白表达工程。表达技术主要使用体内同源重组,采用无缝连接技术克隆PCR扩增的DNA到设计的表达载体中,不需要使用酶或纯化步骤。一管宿主菌可以同时用于稳定克隆和可控的蛋白表达。使鼠李糖表达系统成为最快的有效克隆表达系统。系统工作的完成需要预处理的表达载体和感受态细胞,并需要一次转化过程。

Expressioneering Protein Expression

Figure 1. Expressioneering Technology uses in vivo homologous recombination to seamlessly clone PCR amplified DNA into specially designed expression vectors without the need for enzymes or purification steps. The desired insert is simply amplified with primers that include 18 bases that overlap with the ends of the Expresso® vector. The unpurified PCR amplicon is then mixed with the pRham expression plasmid and the high-efficiency competent cells provided, and directly plated on appropriate media.

鼠李糖表达系统利用鼠李糖诱导型启动子rhaPBAD用于严谨控制蛋白表达。rhaPBAD启动子的转录受不同浓度鼠李糖的控制,能够为困难蛋白的表达找到最适的表达水平。简单的自诱导操作流程是使用盒子自带的鼠李糖和葡萄糖溶液使蛋白表达受到最小的干扰。

pRham™ N-His和 pRham C-His 载体被改系统所提供,可用于克隆靶基因羧基端或氨基端带有6xHis亲和标签。6xHis用于快速简便纯化蛋白,使其保持活性。为了提高蛋白的可溶性,可使用SUMO融合标签技术。见鼠李糖表达SUMO系统。

使用特有的表达技术快速克隆,一个单宿主系统完成克隆和表达,简单的自诱导过程,鼠李糖表达系统适用于高通量的克隆和蛋白表达。 

 
pRham Vectors

Figure 2. pRham expression vectors. RBS, ribosome binding site; ATG, translation start site; Stop, translation end site; Kan, kanamycin resistance gene; ROP, Repressor of Priming (for low copy number); Ori, origin of replication. CloneSmart® transcription terminators (T) prevent transcription into or out of the insert, and a terminator follows the cloning site. The 6xHis affinity tag is fused to the amino terminus (pRham N-His) or at the carboxyl terminus (pRham C-His) of the expressed target protein. Also available: pRham N-His SUMO Vector for enhanced soluble protein expression with cleavable SUMO solubility tag.

 

pRham 表达载体
pRham™ N-His 和pRham C-His 载体被鼠李糖克隆表达系统所提供,如图2.与T7表达盒子的pETite载体相同,鼠李糖表达盒子的载体也是以pSmart为基础改造完成,增加了克隆效率。这是CloneSmart®技术的专利。预处理的pETite和pRham载体的特点是克隆位点两侧都含有相同的序列,允许PCR产物克隆进这两个载体。

使用鼠李糖表达系统单宿主进行克隆和表达

Tuning Recombinant protein expression

Figure 3. Tuning recombinant protein expression levels with rhamnose induction. The pRham C-His Kan Vector containing a gene encoding a blue fluorescent protein (BFP) was transformed into E. cloni 10G cells. An uninduced starter culture was inoculated to a starting OD600 of 0.8 into culture tubes containing LB media with 30 µg/ml kanamycin and the indicated concentrations of rhamnose (0 to 0.2% w/v) or 2% glucose. After overnight incubation at 37°C, samples were harvested by centrifugation, lysed in SDS-PAGE loading buffer, and analyzed by SDS-PAGE. The Coomassie-blue stained gel shows total cellular protein. Protein expression levels are responsive to rhamnose concentrations between 0.001% and 0.2%.

 

rhaPBAD 启动子在缺乏鼠李糖时严谨关闭。使用单宿主进行克隆和表达。诱导水平与鼠李糖浓度
相关(图3)。允许你进行表达水平的调控,尤其是对于毒性和不可溶蛋白的表达。rhaPBAD启
动子的最大量蛋白表达低于T7启动子,但是也能达到较高水平(100 mg/l)。

 
Autoinduction of protein expression

Figure 4. Autoinduction of protein expression with the Expresso Rhamnose System. Flasks of LB media containing 30  µg/ml kanamycin, 0.2% rhamnose, and either  0.05% glucose (early autoinduction, upper panel) or 0.15% glucose (late autoinduction, lower panel) were inoculated to an initial OD600 of 0.4 with an uninduced culture of E. cloni 10G cells harboring the T4 ligase gene in the pRham N-His Vector. Samples of the cultures were harvested at the indicated time points for SDS-PAGE analysis. Induction of ligase expression began by 4 hours in the early autoinduction culture, or 8 hours in the late autoinduction culture. Both cultures reached similar OD600 by 24 hours.

   
Direct autoinduction of recombinant protein

Figure 5. Direct autoinduction of recombinant protein expression from individual colonies. The pRham C-His Vector containing the BFP gene was transformed into E. cloni 10G cells and plated on YT agar plates containing 30 µg/ml kanamycin. Single colonies were picked from the plate and inoculated directly into LB liquid media containing kanamycin (30 µg/ml), rhamnose (0.2% w/v), and either 0.05% (early autoinduction) or 0.15% (late autoinduction) glucose. Samples were harvested at the time points indicated and analyzed by SDS-PAGE.

鼠李糖表达系统便捷的自诱导
rhaPBAD 启动子的转录受到葡萄糖的抑制。当鼠李糖和葡萄糖共存时,葡萄糖代谢优先于鼠李糖,因此启动子是无活性的,当葡萄糖消耗完,启动子被鼠李糖激活。使用葡萄糖和鼠李糖结合方便自诱导程序,可以诱导蛋白表达可以最小化干扰。从一个起始的培养物或者不同的克隆接种进自诱导培养基,诱导自动发生。诱导的时间可以通过使用不同浓度葡萄糖溶液来控制。葡萄糖和鼠李糖溶液盒子自带。

See Application Note in Nature Methods, "Expresso® Cloning and Expression Systems: Expressioneering™ Technology streamlines recombinant protein expression."

Also available: Expresso Rhamnose SUMO System with cleavable 6xHis SUMO tag for enhanced solubility of expressed proteins.

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.

  • Ordering

  • Specifications

  • Manuals

  • Resources

Contact your local distributor for pricing
Product Description Size Cat. No.
Expresso® Rhamnose Cloning and Expression System, C-His   5 rxns 49012-1
    10 rxns 49012-2
Expresso® Rhamnose Cloning and Expression System, N-His   5 rxns 49011-1
    10 rxns 49011-2
Expresso® Rhamnose Cloning and Expression System, N/C-His Combo, 5 reaction of each of N-His and C-His   10 rxns 49010-1
Expresso® Rhamnose SUMO Cloning and Expression System   5 rxns 49013-1
    10 rxns 49013-2
Glucose Solution, 15% w/v   5 x 1.25 ml 49022-1
Rhamnose Solution, 20% w/v   5 x 1.25 ml 49021-1
SUMO Express Protease   200 U 30801-2
鼠李糖克隆表达系统含有预处理的pRham N-His and/o和r pRham C-His 载体 DNA, 单管转化的 E. cloni 10G化学感受态细胞(SOLOs), 自诱导试剂 20%鼠李糖溶液和15%葡萄糖溶液。也含有N-His 或 C-His阳性对照插入DNA,转化阳性对照PUC DNA和鉴定克隆的正反向引物。
鼠李糖 SUMO 克隆和表达系统含有预处理的pRham N-His SUMO 载体DNA, 单管转化的 E. cloni 10G化学感受态细胞(SOLOs), 自诱导试剂 20%鼠李糖溶液和15%葡萄糖溶液。也含有N-His 或 C-His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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