E.coli Cell-Free Expression

E.coli Cell-Free Expression

In vitro protein expression is the use of translation mechanisms of biomolecules extracted from cells to produce recombinant proteins in solution. Because protein synthesis occurs in cell lysates and not in cultured cells, this method is also known as cell-free protein expression. Profacgen has developed a variety of protein expression systems using cell extracts, such as E. coli cell-free expression system, wheat germ cell-free expression system, rabbit reticulocyte lysate cell-free expression system, and tobacco cell-free expression system.

Profacgen offers a variety of E. coli cell-free expression systems, the most commonly used is the E. coli S30 extract expression system. E. coli S30 Extract Systems is prepared by using omp T intracellular protease and lon protease activity mutant mutant strain E. coli B, and achieves the expression of the target protein by adding amino acids, T7 RNA polymerase and energy substances.

Protocal of the E.coli cell-free expression system:

  1. Preparation of E. coli B bacterial liquid, collecting bacteria in logarithmic phase;
  2. E. coli cell disruption (ultrasonic, glass bead grinding, low temperature and high pressure, etc.), prepare E. coli S30 extract system, and add amino acid, T7 RNA polymerase and energy substance to form the reaction system;
  3. Add plasmid DNA or PCR products, synthesize mRNA in vitro under the action of RNA polymerase, incubate and detect;
  4. Use transcription factors in cell extracts, enzymes required for various synthetic proteins, and supplementary amino acids, energy substances, tRNA, etc. to translate mRNA into proteins and purify the target protein;
  5. The mRNA released after translation is recycled using a cell-free system to synthesize the protein, and the mRNA is inactivated after repeated several times.

Advantages of E.coli cell-free expression system:

  1. Extract preparation system is simple and cost-effective;
  2. Convenient and fast expression of target protein, saving a lot of time;
  3. High protein synthesis rate, we can achieve the simultaneous expression of multiple proteins in the same system;
  4. Can be directly used in protein interaction verification experiments and fold complex proteins and complete rapid protein screening. ;
  5. Realize the preparation of labeled proteins and the introduction of special amino acids;
  6. Can realize the expression of transmembrane proteins and solve the problem of prokaryotic expression of some proteins as inclusion bodies;
  7. Can directly interfere with the expression of proteins, to achieve artificial manipulation of protein expression.

Our scientists can optimize the codon preference of specific proteins according to your needs based on the special properties of the protein of interest, construct vectors and express them instantly in E. coli cell-free protein expression systems, and detect specific protein transcription at the RNA level and detect the expression of the target protein.

Profacgen's cell-free expression platform can provide you with comprehensive technical services, specializing in the special properties of the target protein, and professionally solve protein expression related problems, such as low protein production, expression of special proteins (such as membrane proteins, toxic proteins, etc.), and preparation of proteins Complex, parallel synthesis of many different proteins, etc. We always put your needs first, and if you have any questions about the results, we can help you verify. Profacgen is your ideal partner. If you are interested in our services, please feel free to contact us for more information.

INQUIRY

  •   Email: info@profacgen.com
  •   Tel: 1-631-448-8149
  •   Fax: 1-631-938-8127
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