Bacterial Expression Support—Getting Started

Please see the chart below showing the differences in expression, basal expression level without induction (leakiness of expression), and whether the amount of protein expressed can be controlled by the amount of inducer added (titratability). 

To express two proteins at the same time in E. coli, we suggest using a dual promoter vector or using two different but compatible vectors at the same time. For example, you could try a pET vector with a pRSET vector, which contain different ORI (pBR322 origin and pUC origin, respectively). The only issue is that the pRSET vector is high copy number but pET is not; therefore, you may get significantly more protein expression from pRSET than from pET if you add them into one host cell.

Western blot analysis is typically used to detect the expressed protein. We sell several antibodies against various epitopes, such as Xpress™, HisG, V5, or C-terminal 6xHis. Additionally, His-tagged proteins can be purified using our ProBond™ Purification System via affinity purification.

The sequence between the RBS and ATG should be between 8–12 bp and not contain any palindromic sequence.

No; while transcripts will be made, there is no ribosome binding site (RBS) or Shine Dalgarno sequence to initiate translation. mRNA will be transcribed in E. coli cells, but this message will not be translated into protein.

This can vary somewhat, but we typically suggest a starting range of 0.1–5 mM IPTG.  

The time of induction can vary widely. Successful experiments using the T7 systems have induced at an OD₆₀₀ of 0.1–1.2. Generally speaking, induction at high ODs will lead to lower expression yields, as the cells will stop growing rapidly after the density is too high. The optimal OD₆₀₀ for induction is 0.4 to 0.6.

Induction can be performed at a variety of temperatures, ranging from 37°C to 30°C to room temperature. A lower temperature typically requires a longer growth time.

The T7 expression system allows for high-level expression from the strong bacteriophage T7 promoter. The system relies upon the T7 RNA polymerase. While it is not endogenous to bacteria, some strains of E. coli (such as BL21 (DE3) and BL21 (DE3)pLysS) have been engineered to carry the gene encoding for this RNA polymerase in a piece of DNA called the DE3 bacteriophage lambda lysogen. This lambda lysogen contains the lacI gene, the T7 RNA polymerase gene under control of the lacUV5 promoter, and a small portion of the lacZ gene. This lac construct is inserted into the int gene, thus inactivating it. Disruption of the int gene prevents excision of the phage (i.e., lysis) in the absence of helper phage. The lac repressor represses expression of T7 RNA polymerase. Therefore, under normal circumstances in these cells, the lac repressor (the lacI product) binds to the lac operator region between the lacUV5 promoter and the gene encoding for T7 RNA polymerase. This effectively prevents transcription of the T7 RNA polymerase gene. Of course, there is always a small basal level of T7 RNA polymerase present. This is due to the fact that the lac repressor is in equilibrium with the lac operator region, causing the operator site to be occupied most, but not all of the time.

Adding a substance that prevents the lac repressor from binding to the lac operator then induces protein expression. This compound is isopropyl b-D-thiogalactoside (IPTG). IPTG binds to the lac repressor, changing its conformation in such a way that it is no longer able to bind the lac operator. This enables the cells to make T7 RNA polymerase in much more substantial amounts. As the T7 RNA polymerase is specific for the T7 promoter (which is only found in the transformed plasmid), the protein encoded by the plasmid will be overexpressed.

There are several factors that can affect expression including:

• Amount of inducer (IPTG) added
• Time of induction (optimal OD₆₀₀ for induction is 0.4 to 0.6)
• Duration of induction
• Induction temperature
• The construct itself

Transcripts can be made, but there is no ribosome binding site or Shine Dalgarno sequence to initiate translation; therefore, little protein will be produced.

The T7 promoter–based expression systems usually give fairly high yield and can be scaled up easily. Yields will vary depending on the protein being expressed, but in general yields range from 100 µg to 10 mg per liter of culture.

Yes; in fact, carbenicillin is generally more stable than ampicillin and may help to increase expression levels by preventing loss of the pET plasmids.

Both the T7 and Champion™ pET expression vectors contain a strong bacteriophage T7 promoter. After induction with IPTG, T7 RNA polymerase will bind the T7 promoter, leading to transcription and translation of your gene of interest. Studies have shown that there is always some basal expression of T7 RNA polymerase from the lacUV5 promoter in λDE3 lysogens, even in the absence of inducer (Studier and Moffatt, 1986). In general, this is not a problem, but if the gene of interest is toxic to the E. coli host, basal expression of the gene of interest may lead to plasmid instability and/or cell death. To address this problem, the Champion™ pET vectors have been designed to contain a T7lac promoter to drive expression of the gene of interest. The T7lac promoter consists of a lac operator sequence placed downstream of the T7 promoter. The lac operator serves as a binding site for the lac repressor (encoded by the lacI gene) and functions to further repress T7 RNA polymerase–induced basal transcription of the gene of interest in BL21 Star™ (DE3) cells.

We recommend our Champion™ pET SUMO Protein Expression System (Cat. No. K300-01). Clone and express your gene of interest as a fusion to SUMO. Use SUMO Protease to cleave SUMO, resulting in the production of native protein with no extra amino acids added between the cleavage site and the start of your protein.

The pTrc promoter in the pTrc system is a strong hybrid promoter composed of the -35 region of the trp promoter and the -10 region of the lacUV5 promoter/operator. Expression of pTrc is repressed by the LacI protein and induced by IPTG.  

A transcriptional activator protein called CAP (catabolite activator protein) normally binds upstream of the trc promoter and activates transcription. This protein requires cAMP to bind the DNA. Adding glucose to the medium can reduce intracellular cAMP levels. Supplementing LB medium and agar plates with glucose will repress basal level transcription from the trc promoter. We recommend that you include 25 mM, 0.5% glucose in the selection medium to ensure stability of your insert.

The pTrc promoter is recognized by E. coli RNA polymerase, not T7 polymerase, and therefore can be expressed in any E. coli strain, not just BL21 strains. Therefore, you can use Top10, DH5α™, etc. for expression. However, if your gene is toxic, the cloning step can be difficult as there is leakiness in expression.  

The pBAD expression system allows tightly controlled, titratable expression of your protein through the regulation of specific carbon sources such as glucose, glycerol, and arabinose. pBAD is ideal for expressing toxic proteins and optimizing protein solubility in E. coli. The system helps to produce proteins at a level just below the threshold at which they become insoluble. The regulatory elements of the E. coli arabinose operon are used in the pBAD vectors to precisely modulate heterologous expression levels, allowing optimization of the yields of the protein of interest. The regulatory protein, AraC, is provided on the pBAD vector backbone, allowing for regulation of pBAD.

We recommend using L-arabinose to regulate expression of your gene when using the pBAD expression system. In the presence of L-arabinose, expression from PBAD is turned on while the absence of L-arabinose produces very low levels of transcription from PBAD (Lee, 1980; Lee et al., 1987).

The araBAD promoter (pBAD) used to control expression of T7 RNA polymerase in BL21-AI™ is both positively and negatively regulated by the product of the araC gene (Ogden et al., 1980; Schleif, 1992). AraC is a transcriptional regulator that forms a complex with L-arabinose. In the absence of L-arabinose the AraC dimer contacts the O2 and I1 half sites of the araBAD operon, forming a 210 bp DNA loop (see figure below). For maximum transcriptional activation, two events are required.

• L-arabinose binds to AraC and causes the protein to release the O2 site and bind the I2 site that is adjacent to the I1 site. This releases the DNA loop and allows transcription to begin.
• The cAMP activator protein (CAP)-cAMP complex binds to the DNA and stimulates binding of AraC to I1 and I2.

Please note, basal expression levels can be repressed by introducing glucose to the growth medium. Glucose acts by lowering cAMP levels, which in turn decreases the binding of CAP. As cAMP levels are lowered, transcriptional activation is decreased.

The pBAD vectors contain a forward and reverse pBAD primer flanking the gene of interest. The sequences are as follows:

pBAD forward primer: 5’-ATGCCATAGCATTTTTATCC-3’

pBAD reverse primer: 5’-GATTTAATCTGTATCAGG-3’

We recommend using a competent cell strain that is araBADC- and araEFGH+, allowing transportation of L-arabinose, but not metabolizing it. This is important for expression studies, as the level of L-arabinose will be constant inside the cell and will not decrease over time. We offer our TOP10 competent cells, or our LMG194 E. coli strain.

Please review the pros and cons for using TOP10 or LMG194:

While the amount of L-arabinose can vary depending on your expression experiment, we suggest performing a pilot expression experiment with varying amounts of L-arabinose from 0.00002% to 0.2%.

All BL21 cells are derived from strain B834, and are therefore B strains. 

The largest size plasmid we’ve tried is 16 kb, but you should theoretically be able to transform a plasmid as large as 30 kb before efficiency begins to drop.

In all BL21 (DE3) cell lines, there is always some basal level expression of T7 RNA polymerase (note that this is not true for the BL21 AI cell line). If a toxic gene is cloned downstream of the T7 promoter, basal expression of this gene may lead to reduced growth rates, cell death, or plasmid instability. Utilizing a variant cell line that contains a gene encoding the T7 lysozyme as well as the usual DE3 components can circumvent this problem. T7 lysozyme has been shown to bind to T7 RNA polymerase and inhibit transcription. This activity is exploited to reduce basal levels of T7 RNA polymerase. Upon induction with IPTG, the lac repressors no longer bind to the lac operator region and T7 RNA polymerase is produced. This increased level of T7 RNA polymerase production exceeds the limited capacity of the few T7 lysozyme proteins present to inhibit T7 RNA polymerase, resulting in expression of the gene of interest. The BL21-AI cell line can also be used to avoid basal expression with toxic proteins (see below for more details). T7 lysozyme is a bifunctional enzyme. This means that in addition to its T7 RNA polymerase binding activity, it also cleaves a specific bond in the peptidoglycan layer of the E. coli cell wall. This activity increases the ease of cell lysis by freeze-thaw cycles prior to purification.

Minimizing basal expression is particularly important for pET vector expression when hosts that do not carry the pLysS plasmid are allowed to grow to stationary phase (16 hr; overnight cultures) and when the target gene is toxic. Without the T7 lysozyme from the pLysS plasmid, basal expression levels are elevated in cultures grown to stationary phase. If the gene is toxic, the addition of 0.5–1% glucose to both liquid medium and agar plates may be necessary to maintain plasmid stability. Hosts containing pLysS may express an elevated level of lysozyme in cultures grown to stationary phase such that induced levels of the target protein are lowered. 

There are two cell lines that contain the T7 lysozyme; these are called BL21 (DE3) pLysS and BL21 (DE3) pLysE. The following table summarizes some of the key features of the two cell lines.

The BL21 AI E. coli strain offers the tightest regulation of expression for production of toxic proteins using the T7 promoter. The BL21 AI line uses a completely different mechanism of induction from that of the traditional BL21 (DE3) lines. This cell line utilizes an araBAD promoter cloned upstream of T7 RNA polymerase. This replaces the lacUV5 promoter driving the T7 RNA polymerase gene and all but eliminates the leakiness of the traditional BL21 (DE3) expression systems. This eliminates the need for pLysS and pLysE plasmids. In general, the expression yields from this strain are similar to that of other BL21 strains. 

The BL21-AI strain was constructed by inserting a chromosomal copy of the T7 RNA polymerase gene (T7 RNAP) under control of the arabinose-inducible araBAD promoter. The BL21-AI strain does not contain the (DE3) lysogen to be induced by the IPTG. Therefore, you can express proteins from vectors containing the lac/tac promoter.  

BL21 (DE3) Star™ cells contain a mutation in the gene encoding RNase E (rne131), which is one of the primary enzymes involved with mRNA degradation in E. coli. This mutation significantly improves the stability of mRNA transcripts and thus increases protein expression yields over regular T7 promoter–based cell lines like BL21 (DE3). Since T7 RNA polymerase synthesizes mRNA faster than E. coli RNA polymerase; transcription from the T7 promoter is not coupled to translation, leaving a pool of unprotected mRNA transcripts in the cell. These unprotected mRNAs are susceptible to enzymatic degradation by endogenous RNases, greatly reducing protein yield. BL21 Star™ strains contain a mutation in the gene encoding RNase E (rne131), which is one of the primary enzymes involved with mRNA degradation in E. coli. This mutation significantly improves the stability of mRNA transcripts and thus increases protein production.  

We suggest using TOP10 or a similar strain, like DH5α™, for characterization of the fusion, propagation, and maintenance. The presence of T7 polymerase, even at basal levels, can lead to expression of the desired gene even in the absence of inducer. If the gene is toxic to the E. coli host, plasmid instability and/or cell death can result. Additionally, BL21 cells are not endA and recA wild type. This makes them a poor propagation and maintenance host cell line.

Once you have obtained your desired construct, we recommend that you store your clone as a glycerol stock. Please follow these steps to create a glycerol stock:

• Grow 1 to 2 mL of the strain to saturation (12–16 hours; OD₆₀₀ = 1–2) in LB containing 50–100 μg/mL ampicillin
• Combine 0.85 mL of the culture with 0.15 mL of sterile glycerol
• Mix the solution by vortexing
• Transfer to an appropriate vial for freezing and cap
• Freeze in an ethanol/dry ice bath or liquid nitrogen and then transfer to –80°C for long-term storage.

The DE3 designation means the strains contain the lambda DE3 lysogen that carries the gene for T7 RNA polymerase under control of the lacUV5 promoter. This promoter is regulated by the endogenous E. coli lacI protein and is induced with IPTG. IPTG is required to induce expression of the T7 RNA polymerase. The DE3 lambda derivative also contains the immunity region of phage 21.  

BL32-AI™: F- ompT hsdSB (rB-mB-) gal dcm araB::T7RNAP-tetA

The BL21-AI™ strain is an E. coli expression strain that is deficient in the ion protease and outer membrane protease, OmpT. The lack of these proteases reduces degradation of proteins expressed in this strain. The strain carries a chromosomal insertion of a cassette containing the T7 RNA polymerase and tetA genes in the araB locus, allowing expression of the two genes to be regulated by the araBAD promoter. The presence of the tetA gene confers resistance to tetracycline and permits verification of strain identity using tetracycline.

You can assume the protein may be toxic when any of the following occurs:

1.  No colonies are obtained after transformation and growth on plates, or a combination of large and small, irregular colonies appears on the plate.
2. The initial liquid culture does not grow.
3. It takes longer than 5 hours after a 1:20 dilution of the initial culture for the expression culture to reach an AOD₆₀₀ of 0.4.
4. The cells lyse after induction with L-arabinose (or L-arabinose and IPTG).

MagicMedia™ Medium is an E. coli expression medium. It produces 3–10 times higher protein yield, is compatible with T7 regulated E. coli vectors that contain a functional lac operon and BL21 (DE3) strains, is simple to follow, and eliminates OD monitoring/induction steps.  

Unfortunately, MagicMedia™ Medium will not work with the arabinose inducer. MagicMedia™ Medium needs an IPTG-inducible system along with an intact Lac operon.

Yes, expression at 18°C and 25°C has been validated using the Dual Temperature expression protocol.