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8 conditions for microorganisms to express protein

Update time : 2020-09-27

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8 conditions for microorganisms to express protein

Protein expression in microorganisms is a core technology in biotechnology. It is relatively easy to express the protein in a host similar to the source of the target protein. 

However, most of the time, we need to express protein in a host different from the source of the protein, such as expressing eukaryotic proteins in prokaryotic cells, which is usually challenging.

Here, General Biosystems shares with you several tools that help to achieve protein expression across systems.

1. Codon optimization
Codon optimization is to introduce codons that the host system is more willing to read into the vector we constructed, so that it can be translated into amino acids more easily, which will increase the translation speed and increase the efficiency of protein expression.

2. Purification tags

When we express proteins in microorganisms, we usually end up purifying the proteins. Of course, if you only use whole cell extracts or lysed extracts, you don't need to consider protein purification. 

In order to achieve protein purification, we must add purification tags to the protein. At present, many purification tags have been systematically studied and applied in practice. Commonly used purification tags include His tag, Strep-II tag, GST, Flat-Tag, etc.

3. Molecular chaperones and folding enzymes
In order to obtain a useful protein, it is very important to ensure that the protein is folded correctly. In order to help the protein to fold correctly and prevent the formation of insoluble inclusion bodies, we need helper vectors that express chaperones and folding enzymes.

4. Protein expression location

Before the experiment, we need to consider where the protein is expressed, whether it is secreted in the cytoplasm, intercellular substance or extracellular. 

If a protein contains disulfide bonds, we'd better express it in the oxidative environment of the intercellular substance. 

The oxidizing environment can help proteins form the correct disulfide bonds to obtain the correct structure. 

If you want the protein to be expressed at a specific location in the cell or secreted outside the cell, you need to add a signal peptide to the N-terminus of the protein. With the help of signal peptides, proteins can be transferred to specific locations.

5. Protein expression pathway

Protein secretion and expression has many advantages. On the one hand, secreting and expressing proteins can reduce the toxicity and metabolic burden on the host bacteria and increase the adaptability of the bacteria; on the other hand, the host bacteria protein content in the periplasmic space and extracellular medium is very low, which is conducive to the purification of the target protein. 

Different expression pathways have a great influence on the folding efficiency of proteins.

6. Expression strain

Different Escherichia coli strains have great differences in culture conditions and foreign gene expression capabilities. Therefore, the selection of different host bacteria plays a vital role in the accumulation of expression products and downstream separation and purification. 

The use of mutant strains defective in protease expression can increase the secretion and expression of recombinant proteins. 

Some defective strains (such as gene mutants that synthesize outer membrane elements) can produce soluble active proteins with correct spatial structure and disulfide bonds, and It can avoid the hindering effect of the cell wall in the transmembrane transport of foreign proteins and facilitate the secretion of recombinant proteins into the culture medium.

7. Factors such as culture medium, temperature, induction conditions, etc.

The medium composition, culture method, culture conditions and accumulation of inhibitory metabolites during the culture process will affect the expression and secretion of recombinant protein in engineered bacteria. Among them, the nutrient composition of the medium, pH and temperature are important factors that affect the growth and expression of foreign proteins of engineered bacteria, and can affect the activity, secretion and expression of protease. 

The addition of glycine to the culture medium can promote the secretion of protein from the periplasmic space to the extracellular without causing autolysis of the bacteria, and does not cause significant bacterial lysis. Adding sugar gum and TritonX-100 to the medium can prevent the formation of inclusion bodies in the peripheral cavity and improve the efficiency of extracellular expression. 

Changing the osmotic pressure of the medium, short-term thermal shock induction and lowering the temperature during induction will significantly increase the soluble expression of recombinant protein in E. coli. 

In addition, for strains that require IPTG induction, the time of induction, temperature and culture time must be optimized.

8. Cell culture time

Appropriate cell culture time is an important factor that determines cell integrity and protein expression level. Long cell culture time will cause cell lysis, loss of target protein, and release of toxic proteases. 

The direct consequence of too short cell culture time is that the cell concentration is not enough and the target protein yield is too low. Therefore, the cultivation time of recombinant strains must be optimized.