E.coli Expression System Q&A

After years of development, E. coli expression system is a very mature system compared to others. The system has the following characteristics, such as clear genetic background, easy to cultivate and control, simple transformation operation, high expression level, low cost and short turnaround. This article will focus on the common technical problems of E. coli expression system and provide optimal strategies.

E. coli Bacteria

Figure 1 E. coli Bacteria

Q1: What are the advantages of the E. coli expression system?

(1) Bacteria are among the fastest reproducing organisms in the world, doubling every 4 to 20 minutes. It means inoculation (MOI) at a ratio of 1/100, only a few hours would be taken for the bacteria to reach the stable period in the culture medium.

(2) It is easy to realize high-density culture. The theoretical culture density is 1 × 1013 cells/ml. In the actual culture process, LB medium is used to cultivate E. coli at 37°C,<1 × 1010 cells/ml. When recombinant gene expression is induced at low temperature (11℃) , the number of bacteria hardly increases.

(3) It is easy to transfect foreign DNA, and the efficiency of plasmid transfection is high.

Q2: What kind of plasmid system to choose?

At present, the most commonly used recombinant protein expression plasmid vectors are fused with replicon, promoter, selection marker, multiple cloning site (MCS) and fusion tag removal strategy.

Recombinant Protein Expression Plasmid Vector

Figure 2 Recombinant Protein Expression Plasmid Vector

The replicon includes the origin of replication and related cis-acting control elements. When selecting a suitable plasmid vector, the plasmid copy number should be an important parameter to be considered. Several commonly used vector series, such as pET, pUC, pACYC, pBAD and pSC101. The pET series vector contains the pMB1 initiator, which usually contains 15-60 copies in a single bacterial cell. By modifying the pET vector, a dual expression plasmid (BiPlasmid vector) can be obtained, which contains dual MCSs, dual T7 promoters, and dual lac operons and binosome binding sites. The pUC series contains a mutant version of the pMB1 initiator, and the vectors in this series usually have 500-700 copies in a single bacteria. The pACYC and pBAD series are often used in dual expression systems. For example, in the FRET system, this series contains the p15A initiator, and a single cell contains 10-12 copies. The number of copies in a single cell of the pSC101 series vector is usually<5, which is often used for the co-expression of three proteins.

In the E. coli expression system, the lac promoter is one of the most commonly used promoters. When there is only lactose as the sole carbon source in the medium, the lac promoter is activated and the recombinant gene expression begins. The pET series vector with T7 initiation system is commonly used to express the target protein. When the plasmid containing the T7 initiation system is transfected into the bacteria, a single clone is picked and cultured. Then add IPTG (isopropyl-β-D-thiogalactopyranoside, a non-hydrolyzable lactose analogue) to induce bacterial expression of recombinant protein. In order to eliminate the background expression of recombinant protein, you can add the sequences of T7 lysosome and T7 RNAP (RNA polymerase) to the original pET series plasmid vector. Thus, T7 lysosome can bind to T7 RNA polymerase and limit T7 promoter-mediated initial transcription.

The use of affinity tags in the expression of recombinant proteins is to make the protein purification process easier, and promote the solubility of certain insoluble proteins. Protein tags can be divided into two categories: short peptide tags and long peptide tags (co-expressed in the form of fused partner). Long peptide tags are more effective to promote protein solubility, and short peptide tags are often added at the same time to help protein purification. Short peptide tags generally contain only a few amino acids and have a molecular weight less than 2 kDa, which has little effect on the properties of recombinant proteins. Affinity tags can be placed at the C-terminus and N-terminus of the protein. If secreted proteins need to be expressed, tags are recommended to be placed at the C-terminus and the signal peptide at the N-terminus. Different protein tags need to be selected according to specific needs.

Q3: What are the methods to remove protein tags?

There are two types of methods for removing protein tags, one is enzymatic digestion and the other is chemical lysis. Chemical cleavage is usually used for the removal of fusion partner proteins. For example, CNBr is used to cleave peptides connected to the C-terminus of Met amino acids. The advantage of this method is that it is cheap, but the disadvantage is that Met amino acid residues exist in the protein sequence. Enzymatic digestion is currently the most commonly used method of protein label removal, such as enterokinase, thrombin, factor Xa and tobacco etching virus protease (TEV protease) to remove protein labels, leaving only a few amino acids Residues. TEV protease with His tag has gradually been widely accepted for protein tag removal experiments. This protease has an excellent feature: after the tag is removed, only one Ser or Gly residue or no amino acid residue remains.

Q4: What is a suitable protein expression host?

Prokaryotic expression of recombinant proteins often uses BL21 (DE3) and K-12 derivative strains as expression hosts. BL21 strain lacks Lon protease and outer membrane protease OmpT. The main function of Lon protease is to degrade foreign proteins, and the main function of OmpT is to degrade extracellular matrix proteins. At the same time, the BL21 strain has a congenital hsdSB gene mutation, and the strain loses the ability to methylate and degrade the foreign transfected plasmid, so that the plasmid can be passaged and retained in the progeny bacteria (the hsdSB mutant gene is derived from the parent strain B834). The K-12 series of strains have two major advantages. One is that it can promote the formation of protein disulfide bonds in the cytoplasm, which is mainly caused by the mutation of trxB (thioredoxin reductase) gene. The other is the mutation of recA gene, which allows the foreign plasmid to exist stably in the host strain.

Q5: Why does the E. coli expression system have no or low protein expression results?

The toxicity of the protein before and after induction, or the codon usage bias during the expression process may result in no or low expression of the target protein. It is recommended to solve the problem from the following aspects:

1. Control the background expression level:
Lac-based promoter system, add glucose.
Choose glucose as carbon source medium.
Use T7 promoter expression system and plasmid containing T7 lysosome.
Use strict control plasmid to reduce the number of copies.

2. Control the induced expression level:
Adopt adjustable promoters.
Adopt controllable induced strains.
Reduce copy number.
Adopt suitable toxic protein expression strains.
Adopt secretory expression strategies.

3. Optimize the plasmid DNA codons to adapt to the host codon system.

4. Increase biomass: try a new medium, improve aeration conditions and avoid bathing.

Q6: What leads to inclusion body formation?

Inclusion bodies may be formed due to the formation of incorrect disulfide bonds, incorrect folding, production of low-soluble proteins, and lack of post-translational modifications. The following strategies can be adopted to solve these problems.

1) Use E. coli with cytoplasmic oxidative function for secretory expression.
2) Co-expression of molecular chaperones.
3) Supplement the medium containing the compound chaperones and cofactors.
4) Remove the inducer and add fresh medium.
5) Reduce the expression rate by inducing expression at low temperature or adjusting the concentration of inducer.
6) Change the fusion partner protein to promote the expression of soluble protein.
7) Change the expression host, i.g., changing the prokaryotic expression system to a eukaryotic expression system.

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