Bacterial Expression System
Bacterial host is the most often used expression system due to its simple procedure and low cost, making it very popular. Exon BioSystems provides bacterial expression with the following advantage:
- One-step shopping from gene synthesis or gene cloning, expression and purification
- Optimized expression with inducer, temperature and time course.
- High-throughput compatibility
Although the bacterial expression system is most popular for expressing a protein, this system has some intrinsic defects. Many of the eukaryotic expressed proteins are not properly folded, therefore, tend to aggregate into inclusion bodies. Protein folding and unfolding have been extensively studied but the efficiency of in vitro folding is rather limited. If your expressed protein is aggregated into inclusion bodies, laternative expression systems, such as insect expression system and mammalian expression system should be considered.
- Host Strains
- Codon Optimization
- Induction Conditions
Advantage vs. Disadvantage of Bacterial Expression System
Advantage of bacterial cells
- Simple physiology
- Short generation times, as bacteria grow and multiply rapidly
- High yield of product - up to 10 % of mass (low cost)
With B. subtilis and some others, it is possible to induce secretion of a gene product into the surrounding medium. This method is in use in the pharmaceutical industry in the production of hormones such as insulin and human growth hormone.
Disadvantage of bacterial cells
- Activity. The expressed proteins often do not fold properly and so are biologically inactive.
- Toxicity. The synthesized proteins are often toxic to bacteria preventing the cell cultures from reaching high densities. A solution to this problem is to incorporate an inducible promoter, which may be turned on to transcribe the inserted gene after the culture has been grown
- Modification. Lack of enzymes responsible for post-translational modifications (effect on function of proteins), eg if the protein to be expressed is a glycoprotein, there is no apparatus in the bacterium to attach the necessary sugar residues. In this case, mammalian cell expression system is the best choice.
|Overview of E. coli promoter systems useful for heterologous protein production|
|Promoters||expression Level (inductor)||Key features|
|lac promoter||Low level up to middle (IPTG)||Weak, regulated suitable for gene products at very low intracellular level.
Comparatively expensive induction.
|trc and tac promoter||Moderately high (IPTG)||High-level, but lower than T7 system. Regulated expression still possible.
Comparatively expensive induction. High basal level.
|T7 RNA polymerase||Very high (IPTG)||Utilizes T7 RNA polymerase. High-level inducible over expression.
T7lac system for tight control of induction needed for more toxic clones.
Quite expensive induction. Basal level depends on strain used (pLys).
|Phage promoter pL||Moderately high (temperature shift)||Temperature-sensitive host required. Less likelihood of "leaky" un-induced expression. Basal level; high basal level by temperatures below 30°C. No inducer.|
|tetA promoter/operator||Variable from middle to high level (anhydrotetracyclin)||Tight regulation. Independent of metabolic state. Independent of E. coli strain.
Relatively inexpensive inducer. Low basal level.
|PPBAD promoter||Variable from low to high level
|Can fine-tune expression levels in a dose-dependent manner. Tight regulation possible. Low basal level. Inexpensive inducer.|
|PBAD promoter||Variable from low to high level||Tight regulation. Low basal activity. Relatively expensive inducer.|
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Bacterial Strains for Protein Expression
|Bacterial Strain||Origin||Features||Growth Requirements|
|BL21(DE3)||6EMD||DE3 lysogen contains T7 polymerase upon IPTG induction. This strain is defficient of lon and omp-t proteases and is therefore suitable for expression of non-toxic genes.||There is no tight control over the expression.|
|BL21(DE3)-pLysS||EMD||DE3 lysogen expresses T7 polymerase upon IPTG induction. The pLysS plasmid produces T7 lysozyme to reduce basal level expression of the gene of interest. Thus it is suitable for expression of toxic genes.||Chloramphenicol 34ug/ml|
|BL21-AI||Invitrogen||BL21-AI was constructed by inserting a chromosomal copy of the T7 RNA polymerase gene under the tight control of the arabinose-inducible araBAD promoter.||Tetracyclin 12.5ug/ml Expression induction by arabinose.|
|Tuner||Novagen||Contains a mutation in the lac permease (lacZY) gene. This enables adjustable levels of protein expression throughout all cells in a culture.||None|
|Origami||Novagen||Origami host strains are K-12 derivatives that have mutations in both the thioredoxin reductase (trxB) and glutathione reductase (gor) genes, which greatly enhances disulfide bond formation in the cytoplasm.||Kanamycin 15ug/ml Tetracyclin 12.5ug/ml|
|Rosetta||Novagen||These strains supply tRNAs for the codons AUA, AGG, AGA, CUA, CCC, GGA on a compatible chloramphenicol resistant plasmid.||Chloramphenicol 34ug/ml|
|BL21 CodonPlus||Agilent||BL21-CodonPlus-RIL chemically competent cells carry extra copies of the argU, ileY, and leuW tRNA genes. The tRNAs encoded by these genes recognize the AGA/AGG (arginine), AUA (isoleucine), and CUA (leucine) codons, respectively.||Tetracyclin 12.5ug/ml Chloramphenicol 34ug/ml|
|BL21trxB||Novagen||BL21trxB strains possess the same thioredoxin reductase mutation (trxB) as the AD494 strains in the protease deficient BL21 background. The trxB mutation enables cytoplasmic disulfide bond formation.||Kanamycin 15ug/ml|
|C41(DE3)||Lucigen||The strain C41(DE3)was derived from BL21(DE3). This strain has at least one The strain C41(DE3)was derived from BL21(DE3). This strain has at least one uncharacterized mutation that prevents cell death associated with expression of many toxic recombinant proteins. Effective in expressing toxic and membrane proteins from all classes of organisms, including viruses, eubacteria, archaea, yeasts, plants, insects, and mammals.|
It is recommended to compare the codon usage of the bacterial host and the protein of interest: Rare codons such as AGG, AGA, CUA, AUA, CCC, and GGA could be a problem in E. coli. Amino acids may be encoded by more than one codon, and each organism carries its own bias in the usage of the 61 available amino acid codons. In each cell, the tRNA population closely reflects the codon bias of the mRNA. If the mRNA of heterologous target genes is intended to be over expressed in E. coli, differences in the codon usage can impede translation due to the demand for one or more tRNAs that may be rare or lacking in the host. Insufficient tRNA pools can lead to translational stalling, premature translation termination, translation frameshift and amino acid misincorporation. Codon usage optimization and gene synthesis may improve the situation. Synthetic genes are available for less than $1 per base now. It provides the most suitable techniques for codon optimization.
Codon Optimization vs. Codon plus strain?
Beside codon optimization, use of codon plus strains is another way of optimizing codon usage in bacteria. Codon plus strains are transformed for one or two rare tRNA genes. However, the over expression of these genes are usually weak and therefore reduce the ability of prodution of your target protein.
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Protein expression is induced by the addition of the proper inducer or by changing the growth conditions. From this point on the cells will use most of their resources for the production of the target protein and will not grow much further.
- The most commonly used promoters induction conditions are listed below.
|trc (hybrid)||IPTG||0.2 mM||0.05 - 2.0 mM|
|araBAD||l-arabinose||0.2%||0.002 - 0.4 %|
|T7-lac op||IPTG||0.2 mM||0.05 -2.0 mM|
- After induction the cultures are incubated from 3 hours to overnight depending on the induction temperature.
|Incubation temperature||incubation time|
Solubility and Refolding
In E coli, recombinant proteins usually accumulate in the cytoplasm, and examples where recombinant protein constitutes up to 30 percent of total cellular protein can be found in the literature. However, excessive production is not without drawbacks, as the recombinant protein will sometimes misfold and aggregate into so-called inclusion bodies. While inclusion body formation might be advantageous in some cases due to resistance to proteolytic degradation, the subsequent solubilisation and refolding of the inclusion bodies is expensive and results in reduced yield.
Low solubility and inclusion bodies are the main problems when proteins are expressed in the cytoplasm. To circumvent them, you may lower the temperature, substitute amino acids, co-express chaperones, use a large, hydrophilic fusion partner (such as GST), change culture conditions, e.g. pH, or change the bacterial strain. from all these possible approaches, low the temperature from 37°C to 25°C or even 16°C and reduction of inducer IPTG from 1mM to 0.05 mM are used as a routine protocol at Exon BioSystems. Alternatively, inclusion bodies may be solubilized and refolded to get functionality and active products. Inclusion body induction and purification has some advantages, such as high protein induction level, better purification and less protein degradation. The down side is the difficulty of protein refolding. Protein refolding is often inefficiently refolded, and most of the protein will be aggregated. At the same time, the folded protein will have low activity or no activity at all.
Suggestion: avoid protein refolding if possible.
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Glycosylation is perhaps the most common and versatile post-translational modification of proteins. It is estimated that well over one-half of all mammalian proteins are glycosylated. The roles of protein glycans, whether N- or O-linked forms tend to vary. Glycan is required for the biological function of certain proteins, such as,
- The Fc-effector function of immunoglobulin G (IgG).
- The regulated clearance of the glycohormone lutropin (LH),
- The targeting of lysosomal enzymes and,
- Controlling the circulation lifetime of glycoprotein drugs.
Glycans can also serve as:
- Recognition targets for their complementary binding proteins, the lectins.
- Serving as sites of attachment by microbes and toxins, and often constitute the first contact point between pathogen and host.
- Protecting proteins from proteolytic enzymes.
- Promoting protein foldinginto proper tertiary and quaternary structure.
- Modulating the biological activities of protein.
Interestingly, N-glycosylation pathway of baculovirus infected insect cells differs from the pathway found in higher eukaryotes, as indicated by the fact that glycoproteins produced in the baculovirus system typically lack complex biantennary N-linked oligosaccharide side chains containing galactose and terminal sialic acid residues Jarvis DL and Finn EE, Nature Biotechnology 1996).
For authentic glycosylation modification, our mammalian expression systems provides your best choice.
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Commonly Used Tags
|His-tag||6, 8, or 10 aa||Purification||Both native or denature purification.|
|GST||26 kD||Purification||Native purification. Increase solubility.|
|Maltose binding Protein||40 kD||Purification and secretion||Amylose affinity purification|
|Strep II||8 aa||Purification|
|FLAG||8 aa||Purification and detection||Antibody purification, expensive.|
|Protein A||14 kD||Purification||Methotrexate-agrose purification.|
|DHFR||25 kD||Purification||Methotrexate-agrose purification.|
|Cellulose binding Domain||107 aa||Purification and secretion||Cellulose based resin for affinity purification.|
|Calmodulin binding protein||4 kD||Purification and detection||Calmodulin/Ca2+ affinity purification.|
|HA||8 aa||Purification and detection||Antibody purification, expensive.|
|c-myc||22 aa||purification||antibody purification, expensive.|
|NusA||55 kD||Enhance solubility||Potentially improve solubility.|
|Ubiquitin||76 kD||Enhance solubility||Potentially improve solubility|
|GFP||25 kD||Detection||Used as a report gene.|
|HSV||11 aa||Purification||monoclonal antibody-based purification, denaturing low pH elution needed.|