E.coli cell free expression system

The traditional E. coli expression system has been continuously improved and optimized. It has the advantages of short production cycle, high efficiency, low cost and so on. But it also has the defects of the system itself. E. coli expression system can not complete the folding and modification of eukaryotic proteins. So far, this system still can not be used for expressing the membrane protein and some toxic protein expression, which seriously restricts the application of E.coli Expression System. In order to make up for the defects of the prokaryotic expression system, the eukaryotic expression system is commonly used to express the recombinant protein.But the eukaryotic expression system has a series of problems, such as cumbersome procedures, long cycle, low protein production, high cost, and so on. These are not conducive to the popularization and application of eukaryotic expression system. With the deepening of the study of protein expression system, the expression of the protein from the free cell may become a reality. This technique solves the problem that the membrane protein and the toxic protein can not be expressed in the E.coli Expression System, and can only exist in the form of inclusion body.

The E.coli S30 extract expression system is the most common the E.coli cell free expression system. The E. coli B which is OMPT intracellular protease and Lon protease mutant strain is frequently employed to induce the E.coli cell free expression system. This system can be used for the expression of the target protein by adding amino acid, RNA T7 polymerase and energy.

The basic principles of the E.coli cell free expression system are as follows: 1. Directly to the plasmid DNA or PCR product as a template, synthesis mRNA in vitro under the action of RNA polymerase; 2. There include some transcription factors, all kinds of enzymes in extract and add kinds of amino acids, energy substances and tRNA. Under the joint action of these substances, the mRNA is translated into proteins. 3. mRNA is recycled until losing activity.

The basic steps for the E.coli cell free expression system are as follows: 1.E.coli B bacterial liquid preparation and collection of bacteria; 2. E.coli cells were broken and the E. coli S30 extraction system was prepared; 3. Addition of amino acids, RNA T7 polymerase and energy materials was used to prepare the reaction system; 4. Adding expression plasmid, incubation, detection; 5. Protein purification.

Compared with E.coli expression system, E.coli cell free expression system has more advantages. As follows:

1. The direct use of PCR products as template to express the protein can save lots of time;

2. At the same time to achieve expression of multiple proteins in the same system, which can be used for protein interaction experiments directly;

3. It can achieve the simultaneous expression of multiple proteins, complete the rapid screening of protein;

4. Preparation of labeled protein;

5. People can directly regulate the expression of protein.

  The CLOUD-CLONE CROP has been committed to the development and improvement of protein expression system. A large number of protein products which base on the cell free expression system are coming to market. We also accept the protein custom service.

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