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Strategies for high-efficiency E. coli protein expression

Update time : 2020-11-12

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Strategies for high-efficiency E. coli protein expression
The E. coli expression system is the earliest and most mature expression system. Because of its clear genetic background, fast reproduction, low cost, high expression level, easy purification of expression products, good stability, strong anti-pollution ability and wide application range, it is a powerful tool for basic research and commercial production of recombinant proteins; but with this At the same time, the prokaryotic expression system still has many shortcomings that are difficult to overcome: such as the unstable recombinant protein, low biological activity, and expression in the form of inclusion bodies. The expression efficiency of foreign genes in E. coli is affected by many factors. Only by analyzing and optimizing the target protein before expression can the efficient expression of the target protein be achieved, mainly including the following aspects:

(1) The choice of expression vector. 

The choice of expression vector promoter is very important. The structure of the promoter affects its affinity with RNA polymerase, thereby affecting the level of gene expression. The ideal promoter should satisfy:

① It has a strong priming property and can guide efficient transcription to ensure high yield of the target protein;
②It can be strictly regulated, and the expression can be induced under certain conditions, which can minimize the metabolic load of bacteria and the toxic effects of foreign proteins. Others, such as SD sequence position and transcription terminator, will also affect transcription and translation efficiency.

(2) Codon optimization. 

When genes containing a large number of rare codons are expressed in E. coli, the lack of certain tRNAs in E. coli will reduce the efficiency and stability of mRNA translation, and even cause translation errors or premature termination. Codon modification of the target gene can improve the stability and translation efficiency of mRNA.


(3) Fusion proteins and molecular chaperones. 

Fusion proteins can not only be used as affinity tags to facilitate downstream purification operations, but also macromolecular fusion proteins can increase the soluble expression of proteins, such as glutathione-S transferase (GST), SUMO protein , Maltose binding protein (MBP), which is highly hydrophilic, helps to improve the solubility and stability of the target protein; co-expression of molecular chaperones can promote the post-translational folding and processing of recombinant proteins, and improve the solubility and stability of the protein. 

Commonly used in E. coli system are GroEL and GroES systems; heat shock protein DnaK, DnaJ and GrpE ternary system; Dsb family (disulfide bond reductase and isomerase), which can promote the formation of disulfide bonds.


(4) The localization and expression of the target protein. 

After the recombinant protein is synthesized in E. coli, there are 4 localizations, namely in the cytoplasm, periplasmic space, inner or outer membrane, and extracellular matrix. 

Use signal peptides to secrete recombinant proteins into the periplasm or extracellular medium. The oxidative environment in the periplasmic space is conducive to the formation of disulfide bonds and the correct folding of thio-based proteins; the protease in the periplasmic space is lower than that in the cell Many, to reduce the degradation of the target protein; the number of proteins in the periplasmic space is also much less than that in the cell, which is conducive to the purification of the target protein.


(5) Selection of expression strains. 

Too many endogenous proteases in the strain may cause the instability of exogenous expression products. Therefore, some protease-deficient strains often become ideal initial expression strains. 

The Rosetta2 series complements the tRNAs (AUA, AGG, AGA, CUA, CCC, GGA and CGG) corresponding to seven rare codons lacking in E. coli, and improves the expression level of foreign genes, especially eukaryotic genes, in the prokaryotic system; K -12 Derived strains of Origami 2 series, trxB and gor double mutant strains, significantly increase the probability of disulfide bond formation in the cytoplasm and promote soluble protein expression. Rosetta-gami™ combines the advantages of the above two types of strains. 

It not only supplements 7 kinds of rare codons, but also promotes the formation of disulfide bonds and helps express eukaryotic proteins that require the help of disulfide bonds to form a correct folded conformation.


(6) Induction conditions and culture conditions 

Induction conditions and culture conditionsare also critical to the expression of recombinant proteins, such as temperature. When the temperature is higher, the protein expression is high but it is easy to form inclusion bodies, while when the temperature is low, the protein expression is low, but it can increase the target protein Solubility; the concentration of inducer also affects the expression efficiency of recombinant protein, such as low concentration IPTG induction can increase the content of soluble protein. 

In addition, the nutrient composition, pH and other parameters of the medium can affect the activity, secretion and expression level of the protease.



To express foreign proteins in E. coli, analysis before expression is very important. Specific analysis of the target gene and protein, comprehensive consideration, and selection of the most suitable expression system and method can achieve high-efficiency expression of foreign proteins.