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Several effective strategies for protein expression

Update time : 2020-10-28

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Several effective strategies for protein expression
The forum is soaked every day and the experiment is done every day, but the protein is still not expressed well?

The editor hereby presents several effective strategies, which are very effective for obtaining high-yield, functional recombinant protein!

The mammalian expression system overcomes the problem of post-translational modification of protein expression in the prokaryotic expression system. 

The expressed protein has the functions of folding and modification, is highly humanized, and can be used for structural and functional analysis. 

There are usually two ways to express proteins in mammalian cells: transient expression and stable transfection (construction of stable cell lines).


Using stable cell transfection to transfect foreign genes into cell chromosomes can produce the target protein stably for a long time. However, the experiment cost is high, the cycle is long, and the operation is difficult.

Transient protein expression means that the foreign gene exists on the free vector and has not been transfected into the chromosomes of the cell. 

After the plasmid is constructed, the target protein can be obtained through the steps of cell recovery, transfection, cell culture, and protein purification. 

The operation is simple, the experiment cost is low, and the cycle is short. It is most suitable for rapid and small-scale preparation of a certain amount of recombinant protein and recombinant antibody.


Come and see how to improve transient protein expression!

Strategy 1: Optimize the coding sequence

According to the specific downstream application, design the best coding sequence and select the most suitable expression vector. Before starting protein expression, using codon optimization algorithms (such as OptimumGene™ or GenSmart Codon Optimization) and an expression vector selection guide can save time, effort, and expense.
Do you know? GeneralBiosystems can provide integrated services from gene synthesis (free codon optimization) to protein expression and purification!

Strategy 2: Improve protein solubility

Low protein solubility is a key factor that causes low recombinant protein expression yield. We suggest adjusting some commonly used expression condition parameters to improve the solubility of protein.

Temperature:

For HEK293 and other mammalian cell lines, it is recommended to culture cells in a 37°C, 5% CO2 incubator. Some studies have shown that incubating the temperature from 37°C to 33°C 24 hours after transfection can increase the protein expression of HEK293 cells.

Although the basal media provided by different suppliers are very similar, minor differences between them may affect the growth of specific expressing cell lines. 

Be sure to optimize the concentration, pH, type, and even batch or batch number of external components, such as serum added to the culture medium.


Medium additives:

In some cases, we recommend adding specific reagents to the medium to increase protein expression. For example, adding histone deacetylase inhibitors can depolymerize chromatin and increase the transcriptional activity of integrated genes. Alternatively, adding specific growth factors to the medium can help increase protein production.

Strategy 3: Use a fusion partner

Protein tags are short peptide sequences inserted into recombinant proteins and can be used to increase protein expression. It is usually removed by enzyme digestion or chemical reagents at the end of protein expression.

Choosing the right label for a particular expression system depends on many factors. If the goal is to obtain extremely high yields, you need to use solubilizing tags. 

For example, GST has a strong translation initiation signal and can promote high-level expression. However, if a lower expression level is required, it is recommended to use more stringent epitope tags, such as His tags.


Strategy 4: Optimize the purification method

Design different purification methods according to downstream applications and purity requirements. 

If the protein expression level is high, the one-step affinity purification method can be used to obtain sufficient quantity and purity of protein to meet various downstream applications. 

If further purification is required, size exclusion chromatography (SEC) can usually be performed after affinity purification. For further purification, ion exchange chromatography (Ion Exchange, IEX) can be used.