QQ:1443274331

010-88891086,80794780

服务电话:

搜索

 北京市昌平区北清路生命科学园北清创意园2-1-102;  Tel:010-88891086,80794780   Fax:010-80794780 
咨询订购QQ: 1443274331   技术支持QQ: 2074750152   邮件订购:bjzblg@126.com      
www.epibio.com.cn  www.epicentre.cn  www.immunoreagents.cn  www.lucigen.com.cn
版权所有 @北京中北林格科技发展有限公司   
京ICP备:13002510号       网站建设:中企动力 北京

进口试剂

>
>
>
>
HI-Control™ 10G Chemically Competent Cells

进口试剂

HI-Control™ 10G Chemically Competent Cells

浏览量
产品名称:

HI-Control™ 10G Chemically Competent Cells

货号
60110-1
规格
12 rxns (SOLOs)
存储温度
-80º C
没有此类产品
产品描述

Expresso® SUMO Cloning and Expression Systems

 

产品说明书

 

Soluble protein has never been this fast and easy!

  • Enzyme-free directional PCR cloning in seconds!
  • Save days of effort with ready-to-use vectors and competent cells - NO ligation step.
  • Tightly-controlled expression of N-terminal 6xHis-tagged proteins with cleavable SUMO solubility tag.
  • Systems available with expression under the control of T7 or tunable Rhamnose promoters.
  • Expressioneering™ Technology:overview of Expresso® product line featuring ligase-free cloning  
 

Complete Cloning and Protein Expression System

The Expresso SUMO Cloning and Protein Expression Systems are designed for fast, easy, and efficient directional cloning and soluble expression of PCR-amplified genes. For proteins that form inclusion bodies or are difficult to express in soluble form, the pETite® N-His SUMO and pRham™ N-His SUMO Vectors allow expression of target proteins with an amino-terminal 6xHis-SUMO protein tag. SUMO (small ubiquitin-like modifier) is a relatively small (100-residue) polypeptide that has been shown to enhance the soluble expression of many proteins that are otherwise difficult to produce in E. coli.

Comparison Expresso and TraditionalThe 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.

Five-Second Directional Cloning of PCR-Amplified Genes

The Expresso SUMO Cloning and Protein Expression Systems use Expressioneering Technology, the enzyme-free recombinational cloning strategy of the Expresso system to seamlessly integrate the gene with the expression plasmid. The target gene is amplified by PCR using primers that add 18 base-pairs of vector-complementary sequence to both ends of the gene. Unlike other restriction enzyme based methods or ligase-free cloning methods, no further cleanup or enzymatic treatment of the PCR product is necessary. Simply mix 1 µl of the unpurified PCR reaction with the supplied pre-processed pETite or pRham N-His SUMO expression plasmids, and transform immediately into the Chemically Competent Cells provided (Figure 1). 

 

SUMO Vectors includes:

  1. Strong T7 promoter for high-level, or Rhamnose promoter for tunable, recombinant protein expression.

  2. Increased solubility and fast protein purification from N-terminal 6xHis SUMO fusion tag

  3. Small size (2.5 kb) for easier downstream manipulation.

  4. Patented CloneSmart® technology increases cloning efficiency.

     

SUMO Vector Map

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.

Rescue inclusion bodies and insoluble protein with SUMO protein tag:

We have used the Expresso T7 and Expresso T7 SUMO Cloning and Expression Systems for expression and purification of a variety of proteins. Some results of an ongoing large-scale expression study to identify hydrolase enzymes from Fibrobacter succinogenes are presented in Figure 3. Initially, 48 genes were selected for expression trials and cloned into the pETite T7 C-His Vector. Approximately half of these clones have produced soluble, active hydrolase protein, while in other instances target proteins were expressed in an insoluble form. Five of the genes producing insoluble proteins were reamplified and cloned into the pETite SUMO vector. When the resulting clones were expressed in HI-Control BL21(DE3) cells, recovery of active protein in the soluble fraction was significantly improved in four of the five cases (Table 1). Although tag removal was not necessary for hydrolase activity, the tag could be removed efficiently by SUMO Express Protease.

 

Expresso SUMO

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.

 

Fibrobacter succinogenes
gene number

Soluble protein yield

w/o SUMO tag

w/SUMO tag

1425

0 mg/liter

0 mg/liter

1765

0 mg/liter

10 mg/liter

1793

0 mg/liter

17 mg/liter

1994

0 mg/liter

17 mg/liter

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.

Cleavage of SUMO protein tag

After IMAC purification of the N-His-SUMO tagged protein, the tag portion can be removed precisely by the included SUMO Express Protease. The SUMO Express Protease recognizes the tertiary structure of SUMO rather than a short recognition sequence and cleaves precisely at the junction between the SUMO tag and the target protein, with no off-target cleavage. Both the SUMO Express Protease, which is 6xHis tagged, and the cleaved N-His-SUMO tag can then be separated from the released target protein by subtractive IMAC.

Note: The SUMO protein tag included in these kits is a specially engineered version of SUMO that can be cleaved only using Lucigen's SUMO Express Protease. SUMO Express Protease does not cleave the wild type SUMO substrate at any useful level. Therefore, it is not recommended to use SUMO Expresso Protease to cleave wild type 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.

  • Ordering

  • Specifications

  • Manuals

  • Resources

Contact your local distributor for pricing
Product Description Size Cat. No.
Expresso® Biotin SUMO C-Biotin System   5 rxns 49044-1
    10 rxns 49044-2
Expresso® Biotin SUMO N-Biotin System   5 rxns 49043-1
    10 rxns 49043-2
Expresso® Rhamnose SUMO Cloning and Expression System   5 rxns 49013-1
    10 rxns 49013-2
Expresso® T7 SUMO Cloning and Expression System   5 rxns 49003-1
    10 rxns 49003-2
Arabinose Solution, 1000X   5 x 1.0 ml 49023-1
Glucose Solution, 15% w/v   5 x 1.25 ml 49022-1
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
Rhamnose Solution, 20% w/v   5 x 1.25 ml 49021-1
SUMO Express Protease   200 U 30801-2
ORDER INFORMATION

The Expresso T7 SUMO Cloning and Expression System contains pre-processed pETite® N-His SUMO Vector DNA, HI-Control™ 10G Chemically Competent Cells for cloning, and HI Control BL21(DE3) Chemically Competent Cells for protein expression. Also included are SUMO Positive Control C Insert DNA, transformation positive control pUC DNA, SUMO Express Protease, SUMO Cleavage Control Protein, and forward and reverse PCR primers to confirm clones.

The Expresso Rhamnose SUMO Cloning and Expression System contains pre-processed pRham N-His SUMO Vector DNA, single-transformation E. cloni 10G Chemically Competent Cells (SOLOs), and the auto-induction reagents 20% Rhamnose solution and 15% Glucose solution. Also included are SUMO Positive Control C Insert DNA, transformation positive control pUC DNA, SUMO Express Protease, SUMO Cleavage Control Protein, and forward and reverse PCR primers to confirm clones.

未找到相应参数组,请于后台属性模板中添加
暂未实现,敬请期待
暂未实现,敬请期待
上一篇
下一篇