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Several forms of protein expression in E. coli

Update time : 2020-10-19

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Several forms of protein expression in E. coli

It is an important part of molecular biology research to study the structure and function of the foreign gene in the protein expression system in vitro. Protein expression in vitro includes fusion expression and non-fusion expression, as well as expression in mammalian expression systems, yeast expression systems, and insect cell expression systems. Escherichia coli has the characteristics of thorough understanding of hereditary traits, fast growth, economical culture, and high expression level. 

Therefore, E. coli expression has been the preferred expression system for expressing foreign proteins for a long time.



The Escherichia coli expression system is the preferred engineering bacteria for protein expression, and it is also the most widely used classical prokaryotic expression system.

General Biosystems researchers have established a mature E. coli protein expression and purification service platform, which provides expression and purification services including various recombinant proteins and their complexes in E. coli.

Escherichia coli is divided into three cavities by the outer and inner membranes. The target protein expressed by E. coli is located in the three cavities: intracellular, extracellular and periplasm.
According to the location of the expression product, the expression of the target gene in E. coli is generally divided into intracellular expression and secretory expression.
Among them, intracellular expression is the most important form of expression, and its expression products can be divided into soluble expression and insoluble inclusion body forms. According to the nature of the target protein, the expression form of the target protein in E. coli is divided into fusion expression and non-fusion expression.

1. Intracellular expression


The target protein expressed by E. coli is often localized in the cell. In order to avoid excessive accumulation of the expression product in the cell and affect cell growth or form inclusion bodies, the soluble target protein can use its own functional sequence and the processing and transport system of E. coli protein , Finally traverse the cell membrane and secrete into the periplasmic space between the cell membrane and the cell wall or the cell culture fluid.

2. Secreted expression


The target recombinant protein crosses the inner membrane of E. coli to reach the periplasmic space, or crosses the outer membrane to reach the extracellular space, which is called secretion. A series of Sec protein sequence structures related to the secretion of E. coli proteins into the periplasmic space and transmembrane transport outside the cell have been found, including SecA, SecB, SecD, SecE, SecF, SecG and SecY [9, these 7]. In addition to Sec protein, molecular chaperones such as GroES/GroEL, DnaK/DnaJ and h/Ffs are also involved in transport.

3. Soluble expression


The expression of the target gene in Escherichia coli is usually capable of obtaining soluble expression with biological activity. However, foreign proteins are often easily degraded by host proteases or form inclusion bodies while obtaining high levels of expression. 

Therefore, it is necessary to explore the soluble expression of foreign proteins in E. coli. To improve the soluble expression of recombinant protein, it is first necessary to select a suitable vector and host bacteria, and secondly, it can also reduce the rate of recombinant protein synthesis.


Studies on protein kinetics models show that the yield of active protein depends on the rate of protein synthesis, the rate of protein folding, and the rate of protein aggregation. 

At high levels of expression, once the aggregation rate of the nascent peptide chain exceeds the rate of protein folding, it will lead to the formation of inclusion bodies. 

Therefore, reducing the rate of recombinant protein synthesis is beneficial to increase the soluble expression of recombinant protein. 

Commonly used methods include lowering the culture temperature, selecting an appropriate promoter, and using a lower concentration of inducer.



The composition of the medium has a significant effect on improving the soluble expression of recombinant proteins in E. coli. 

Molecular chaperone is a kind of protein that can help the polypeptide chain to form the correct three-dimensional structure. 

It mainly helps the folding of the peptide chain by preventing or correcting unreasonable hydrophobic binding. Studies have found that co-expression with molecular chaperones can improve the solubility of the target protein, and if several molecular chaperones are expressed at the same time, it is possible to obtain better results than using them alone. 

It is worth mentioning that the choice of co-expression molecular chaperones should be based on the characteristics of the recombinant protein itself. 

The primary structure of protein, namely amino acid sequence, is an important factor affecting the formation of inclusion bodies. Replacing one or several amino acids in the protein can inhibit the formation of inclusion bodies and increase the solubility of the expressed product. 

The possible reason is that the hydrophobicity of the recombinant protein changes or the stability of the recombinant protein increases after the mutation.


4. Form inclusion body expression


However, when the target protein is expressed in large quantities, the expression product will accumulate in E. coli, and inactive solid particles will be formed, called inclusion bodies. 

The target protein of the inclusion body has the correct amino acid sequence, but because it is not correctly folded and has a spatial conformation error, it is generally not biologically active. 

Compared with soluble target protein products, inclusion bodies have the advantages of enrichment of target protein, resistance to proteases, and less toxicity to the host. 

The main reason for the formation of inclusion bodies is the lack of some cofactors for protein folding during the expression of the recombinant target protein, or the environment is not suitable for forming correct secondary bonds. 

The main factors affecting the formation of inclusion bodies are the nature of the protein itself, culture conditions, and molecular chaperone proteins.


5. Fusion protein expression


Fusion protein expression is to connect the coding regions of two or more genes end to end, and then controlled by the same regulatory sequence, so that the target protein fusion expression has high efficiency, less degradation, and simple and convenient purification. 

In addition, the chemical reagent cleavage site or protease cleavage site constructed in the fusion expression vector can be used to remove the prokaryotic peptide of the fusion protein in vitro to obtain a natural protein product. 

Because the fusion protein is easy to prepare, it has a higher probability to maintain the activity and immunogenicity of the original protein, and it can also be commercialized to provide the possibility for subsequent cleavage of the fusion protein to obtain the eukaryotic protein inside. 

Moreover, fusion expression can not only increase the amount of protein expression, but also help the target protein to fold correctly to obtain soluble protein.


Currently, the more widely used fusion expression systems include PA system, PG system, His fusion system, GST system, FLAG system, MBP system, TrxA fusion system, etc. 

The expression product expressed in prokaryotic expression is highly efficient, relatively stable, and not easily degraded by the host.


6. Non-fusion protein expression


The non-fusion expression of an expressed protein that is not fused with any protein or polypeptide of the host cell is to insert the target gene into the strong promoter of the expression vector and downstream of the SD sequence, with the initiation codon AUG of the target gene as the starting point of translation. 

The N-terminus of the expressed target protein peptide chain does not contain the protein expressed by the prokaryotic expression vector, and the expressed target protein is consistent with the natural protein in sequence, structure, and immunogen. 

Non-fusion proteins are easily degraded or destroyed by proteases in host cells to produce inactive proteins, which affects expression efficiency.


The expression of foreign proteins in Escherichia coli has the advantages of high expression level, easy operation and low cost. 

Common expression forms include intracellular expression, secreted expression, soluble expression, insoluble inclusion body form, and fusion expression and non-fusion expression. 

Several forms. The expression of foreign proteins in E. coli is a systematic project. Therefore, we should accumulate experience in the research process to find out the key to improving the efficiency of recombinant protein expression. 

Therefore, it is possible to use the E. coli expression system to express large amounts of active foreign proteins.


General Biosystems, a protein expression and purification service company, can provide customers with more common host services, including E. coli, yeast and mammalian cells. If you want to use other host cells or cell-free expression systems, please contact us.