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CherryExpress

Sharpened tools for life-science discoeries

The CherryTMExpress kit allows direct visualization (by eye!) of your protein of interest during protein production in E. coli and protein purification. No special requirements or reagents are needed. It is also
possible to quantify the protein concentration at any step by spectral measurement. The CherryTMExpress kit combines multiple advantages: protein visualization, T7 expression and plasmid stabilization.  See the movie: PC orMAC version

Principle

The Cherry tag vectors allow direct visualization of your protein of interest during the whole process of protein production and purification without the need for any special requirements or reagents. The Cherry tag was incorporated into the StabyExpressTM T7 vector allowing high-level protein expression with or without antibiotics (click here for more info on theStabyTM technology). When using the Cherry tag vectors, your gene of interest is fused to a small sequence encoding a red polypeptide (heme binding part of cytochrome, 11 kDa) providing a visual aid for estimating expression level and solubility: bacteria expressing the fusion protein are red when the fused protein is soluble. The red color also constitutes a visual marker throughout protein purification. Concentration of the fusion protein can be determined easily by spectral measurement (before and after purification). In addition, the Cherry tag is highly soluble and thus, can increase the solubility of target proteins (for more information about protein solubility using CherryTM kits, consult this application note). When using the Cherry tag as a N-terminal tag, it can be cleaved after purification using enterokinase (a recognition site is inserted at the C-terminal end of the tag sequence).
The CherryTMExpress system is compatible with the use of an auto-inducible medium asStabyTMSwitch.

Results

Using the Cherry tag vectors, recombinant protein expression is first detectable from the colorof the bacterial pellet: the bacteria expressing a soluble fused protein are red. As lack of solubility is a major problem when expressing recombinant protein in E. coli, the Cherry tagsystem is convenient for rapid screening and optimization of protein solubility. The tag itself being highly soluble, it can increase the solubility of the target protein.

tubes2_Cherry-1.jpg

Furthermore, it is easy to visualize binding of the protein to the column (affinity or ion exchange) and to verify the absence of remaining protein of interest in the effluent. During elution, it is not necessary to collect multiple fractions and to analyse it to localize the target protein. Indeed, the Cherry tag system allows you to collect only the fraction containing the protein of interest. 

threestepscherry-1.jpg

When using the Cherry tag system, it is possible to quantify the protein concentration at any step (from protein production to the end of purification): a simple absorbance measurement at 413nm allows specific and accurate calculation of the target protein concentration.

Kit Content

The Cherry tag was inserted in the StabyExpressTM T7  vector to yield CherryExpress kit combining multiple advantages: T7 expression, plasmid stabilization and protein visualization. Different types of CherryTMExpress kits are available: cherry tag as N-terminal (pSCherry1 plamsid) or C-terminal tag (pSCherry3 plasmid), kit containing either electrocompetent cells or chemically-competent cells. All kits contain DNA of the vector (pSCherry1 or pSCherry3), DNA of the pStaby1.2 vector, chemically- or electro- competent bacteria for cloning (CYS21) and expression (SE1), regeneration medium, sequencing primers, and the instruction manual. Additional CYS21 or SE1 competent bacteria can be purchased separately.

_pSCherry1_map.jpg
pScherry 1 vector for Cherry N-terminal tag

pSCherry3_mapb.jpg
pSCherry3 vector for Cherry C-terminal tag



Benefits of the CherryTM Tag system

  1. Direct visualization of the protein of interest;
  2. No need of any special apparatus or reagents;
  3. Rapid screening and optimization of protein expression and solubility;
  4. Easy quantification of the protein of interest;
  5. High yield of protein production using StabyTM systems with or without antibiotics

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