Gene Expression

Prokaryotic expression systems FAQs


Q: Low or no protein expression

A:

1, Choose correct expression vectors and strains.

· T7 promoter-based expression vectors (like pET vector series) should be paired with strains like BL21 (DE3), BL21 (DE3) pLysS and Transetta (DE3). For non-T7 promoter-based expression vectors (e.g. pGEX and pMAL vector series with Tac promoter) should be paired with the BL21 expression strain.

· For toxic proteins, it is recommended to use strains with low background expression and strict regulation such as the BL21 (DE3) pLys S strain.

· For proteins with rare codons or proteins from eukaryotic genes, strains such as Transetta (DE3) are recommended.

2, Try different expression vectors and strains

Different proteins have different preferences for vectors and strains. If protein expression cannot be significantly improved by optimizing induction expression conditions, it is recommended to change strains or expression vectors.

3, Optimization of expression conditions

· Choose different media. For some proteins, a proper amount of glucose (0.1%-0.5%) addition can significantly increase the amount of expression.

· Higher temperature, higher inducer concentration, and longer expression time can generally speed up expression and promote the accumulation of target proteins, and thereby increase the amount of expression. However, the solubility may be reduced leading to the formation of inclusion bodies.

· The growth status of cells has a significant impact on protein expression and can be monitored by measuring the OD600 value of the bacteria solution. For most proteins, induction should be performed in llogarithmic growth phase (OD600=0.5) of the strain.


Q: Target proteins are insoluble with the formation of inclusion bodies

A:

Firstly, confirm whether the target protein has been expressed.

· After the lysis of the bacteria and centrifugation, confirm whether the target protein has been expressed and whether the inclusion body has been formed by detection of whole bacteria, supernatant and precipitation.

· The formation of the inclusion body is closely related to the protein's structure, expression system, induced expression conditions and other factors. In the case that the protein's structure cannot be changed, different expression vectors or strains can be tried to enhance protein solubility and optimize for an expression system that is suitable for a specific protein.

· It is currently acknowledged that the formation of the inclusion body is due to too rapid protein accumulation in the cell, thus the protein cannot be correctly folded resulting in protein aggregation and precipitation. The accumulation of proteins can be slowed down by optimizing induced expression conditions, such as reducing induction temperature, reducing the concentration of the inducer, shortening expression time and reducing the OD value of the bacteria solution in induction.


Q: Incorrect target protein size

A:

· Determining molecular weight is affected by the structure of the protein. The molecular weight can be accurately determined by heating the protein to denatured form.

· Confirm if protein expression is complete and early termination has occurred.

· If dimers or even multimers are formed, the molecular weight can be accurately determined by heating the protein to denatured form, breaking disulfide bonds (Add DTT, β-ME or other reducing agents) or other methods that can destroy the secondary structure of the protein.


pEASY Prokaryotic Expression Vectors
pEASY®-Blunt E1 Expression Kit
Expression Competent Cells
BL21(DE3) Chemically Competent Cell
BL21(DE3) pLysS Chemically Competent Cell
Transetta(DE3) Chemically Competent Cell
TransB(DE3) Chemically Comptent Cell
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