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Tuesday, January 3, 2012

Five steps involved in the Expression and Purification of Proteins in E. coli

Production and detailed characterization of a variety of proteins is facilitated by their heterologous expression and purification. Recent advances in genomics have led to a massive increase in the number of proteins being produced using recombinant DNA technology.

In order to express heterologous proteins, a variety of expression systems have been developed. For example, bacteria (e.g. Escherichia coli, Bacillus subtilis, etc), yeasts (e.g. Saccharomyces cerevisiae, Pichia pastoris, Yarrowia lipolytica, etc), filamentous fungi (e.g. Aspergillus nidulance, Trichoderma reesei, etc), insects, plant cell cultures and mammalian cell lines.  
Generally, Escherichia coli is the most commonly used bacterial expression system for expression of the heterologous proteins. Reasons behind this include:

  • Genetic manipulation is easy,
  • Its culturing is inexpensive,
  • Expression is fast,
  • In many cases, the level of expression is high,
  • Majority of foreign proteins are well tolerated, etc.
The above mentioned advantages and many more have guaranteed that E.coli remain a valuable organism for the high-level production of heterologous proteins.

In order to carryout expression of your gene of interest in E.coli, you should follow these steps:

Choosing the Expression vector

Choosing a suitable bacterial expression vector is the first step in the expression of heterologous protein in E. coli. The choice of vector system mainly depends on two factors. These include: i) Transcriptional and translational regulators, and ii) affinity tag.

i) Transcriptional and translational regulators

These elements consist of a set of genetic components that affect both the transcription as well as translation of the heterelogous protein. These elements include:

· Promoter

Promoter is the most important transcriptional element of an expression vector. Its main function is to allow RNA polymerase to bind to the DNA. Therefore, it regulates the rate of mRNA transcription. As a result of this, the amount of heterologous protein produced, largely depends on the type of promoter used.

The promoter which results in high level of transcription is called as strong promoter. However, weak promoter is the one which allows very low level of transcription. Consequently, an expression vector should have strong promoter to carry out highest level of transcription of the cloned gene. The commonly used strong promoters in bacterial expression vectors are, lac, trp, tac, and T7 promoters.

· Repressor

Repressors are the elements which allow the repression of a promoter. When a repressor is bound to the promoter no transcription occurs. Therefore, repressor works as a regulator of promoter activity.

It is very helpful if the protein of interest is toxic to the bacteria. By the use of repressor the accumulation of toxic level of a protein is monitored. Regulation of gene expression is achieved by the calculated use of the chemical.

· Terminator

The expression vector should have both transcriptional as well as translational terminators. A transcription terminator enhances the mRNA stability, therefore, leading to substantially increased level of protein production. In addition, it also stabilizes the plasmid by checking the expression of ROP protein, which is involved in the control of copy number of the plasmid.

Translational termination of the protein is carried out by the presence of a stop codon. Bacterial expression vectors contain stop codons in all the three open reading frames, in order to prevent ribosome skipping.

· Translational initiator

Translational initiation is determined mainly by the unique structural features at the 5’ end of the mRNA. This includes the ribosome binding site (RBS). Generally, the ribosome binding site consists of Shine-Dalgarno (SD) sequence followed by an AT rich translational spacer. These elements establish the translational efficiency of the mRNA.

ii) Affinity Tag

With the advancement of gene cloning methodologies, it is now possible to construct the fusion proteins. In this strategy, specific affinity tags are added to the protein sequence of interest. Addition of affinity tag to the protein of interest, simplifies the purification of target proteins by using affinity chromatography techniques.

Apart from this, use of affinity tags has the following advantages:

· It enhances the efficiency of translation of the target mRNA.
· It protects the target protein from proteolytic degradation.
· Some of the affinity tags (e.g. maltose binding protein or MBP) help in the solubilization of the target protein, hence target proteins remain in the cytoplasm rather than inclusion bodies.

The most commonly used affinity tags are:

His-tag which bind to the immobilized metal ions (e.g. Ni2+).
GST-tag which binds to the glutathione–Sepharose resin.
MBP-tag which binds to the amylose resin.

Cloning the gene of interest

After choosing the suitable expression vector, the next step is to clone your gene of interest. Majority of the expression vectors provide multiple cloning sites (MCS) for the ease of cloning of the gene of interest. Generally, the MCS is placed either between the RBS and the affinity tags or between the affinity tag and the terminator. Therefore, the cloning results in the fusion of affinity tag to N or C-terminal of the target protein, respectively.

However, care should be taken in checking out the frame of the fusion protein. For example, if you are doing a N-terminal fusion, then your sequence of interest should have stop codon. Similarly, if you are doing a C-terminal fusion, then you should maintain the open reading frame with the affinity tag and your sequence of interest should have the start codon (ATG).

After doing the transformation in E.coli, it is always better to confirm cloning by sequencing. You should compare the sequences in order to find out any frame shift mutation or a miss match. These anomalies can result in the incorporation of stop codons thereby leading to premature termination of your target protein.

Expressing the recombinant protein in E.coli

After confirmation of the cloning, the next step is to transform the recombinant plasmid into the E.coli host for protein expression. You cannot use the DH5? or similar strains for expressing your protein. For the expression of heterelogous proteins, certain protease deficient strains of E. coli have been developed. The most widely used strain is the BL21 (DE3).

Transform one vial of BL21 competent cells and select them on plates having the appropriate antibiotics. It is always good to prepare a glycerol stock of a single colony. If you are checking for the first time, then you should prepare stocks of 4-5 colonies separately. It is not a good practice to store the plate at 4°C and use it in future for expression studies. Either you prepare a stock from freshly transformed E.coli or do a fresh transformation. It is observed that these strategies improve the expression of your protein of interest.

For checking the expression you can start with a culture as small as 1ml. LB is the preferred media for the expression of a wide variety of proteins. You will have to optimize the growth conditions. Different proteins are expressed in different growth conditions. These include: temperature, aeration, size of the culture, culture media, concentration of inducer, etc.

Always remember to take a culture having blank vector along with your recombinant plasmid. This will work as a negative control. If you are using an inducible system, then you should also include the un-induced sample.

After doing all these stuffs, simply run the samples on SDS-PAGE along with the protein molecular weight marker. Meanwhile calculate the molecular weight of your recombinant protein (i.e. weight of your protein of interest + weight of the affinity tag). This will help you in examining the protein gel. If you got the band of desired size in the gel, congratulations! You have done it.
Unfortunately, if you are unable to find the band of desired size, check out the above written parameters or take another colony.
It is always good to check the solubility of the recombinant protein. Confirm that the protein is soluble by loading the clear lysate along with the cell pellet. If your protein is soluble then major fraction should be visible in the clear lysate as compared to the cell pellet.

Isolating the protein

After the confirmation of expression of your protein of interest in the soluble fraction the next step is to isolate the protein. For this, the cells are harvested through centrifugation. The next step is to disrupt the cells. Various methodologies have been developed for bacterial cell disruption. The most commonly used are treatment of cells with lysozyme followed by sonication.

Generally, the harvested cells are re-suspended in the lysis buffer. The lysis buffer contains lysozyme at a concentration of around 1mg/ml. Apart from this, certain protease inhibitors like PMSF are also included. The suspension is incubated at 4°C for about half an hour. Then it is gently agitated for nearly 10 min. After this, sonication is carried out. Care should be taken while sonicating the samples. Excessive sonication leads to the denaturing of the target protein.

After disrupting the cells, the suspension is centrifuged at low rpm for around half an hour to separate the lysate from the cell debris. The supernatant obtained after centrifugation is generally called as the cleared lysate. This lysate contains your protein of interest. Now you can proceed for the purification of the protein.

Purifying the protein

After getting the clear lysate the last step is to purify the protein, exploiting the affinity tag used. Depending on the fusion tag used, a suitable resin is chosen for the purification process.

The clear lysate is first allowed to bind to the resin. This is accomplished by mixing the clear lysate along with the resin and gently agitating them for around 30 min. After this the suspension is transferred to a polypropylene column. The suspension is allowed to settle down. After this, the cap of the column is opened and lysate is allowed to pass through the resin.

The next step involves the washing of the resin with appropriate wash buffer. Care should be taken in adjusting the stringency of washing. Higher stringency will lead to the loss of the protein of interest, whereas, lower stringency will cause purification of not target proteins. Therefore, stringency should be carefully adjusted in order to get large amount of target protein with no contamination.

After washing, the protein of interest is obtained by eluting it with the appropriate elution buffer. The composition of the elution buffer depends on the fusion tag used. The isolated protein can be stored at 4°C for shorter duration (around a week) otherwise it should be stored at -20 or -80°C.

This was just the overview of steps involved in the expression and purification of proteins in E.coli. We will be publishing the detailed reviews of each step in future articles. Please feel free to ask any questions by posting it in the comments.


  1. Very usefull information. Thank you....

  2. Really good site. I would appreciate if there were any references.