Modern Biotechnology 2011 (Synthetic biology) - Science of the Unthinkable.!!

Three commonly used affinity tags for protein purification

In order to purify heterologous proteins from various hosts, affinity tags are extremely capable tools. In the recent years affinity tags have become highly popular tools for protein purification mainly due to following reasons. They provide high level of purification of recombinant proteins from crude extracts, mostly in a single step. Second, they provide mild elution conditions, thereby, do not interfere with the structure and hence the function of the purified proteins. In addition, affinity tags allow a variety of proteins to be purified using easy procedures.

Affinity tags are available as expression vector systems having multiple cloning sites (MCS) for cloning the gene of interest towards the N or C-terminal of the tag. Now-a-days, a variety of affinity tags are available for the purification of recombinant proteins. Each tag has its certain advantages and disadvantages. Here, you will see the properties of the three commonly used affinity tags: His-Tag, GST-Tag and MBP-Tag.

1)  His-Tag

His-tag is the most commonly used affinity tag for the purification of recombinant proteins in E.coli. The His-tag is a peptide motif that consists of six histidine (His) residues. Therefore, it is also called as hexa histidine-tag or 6xHis-tag. The most commonly used bacterial expression vector system for His-tag is the pET series (Novagen). These vectors provide both the N & C-terminal fusion of gene of interest with the His-tag.

It is observed that hexahistidine has high affinity towards transition metals e.g. Ni⁺⁺, Co⁺⁺ etc. Therefore, in order to purify recombinant proteins with hexa histidine-tag, immobilized-metal affinity chromatography (IMAC) columns are used. Ni-NTA agarose is the most commonly used resin for the purification of His-tag proteins. Since very few naturally occurring proteins bind to the Ni-NTA matrices with considerable affinities, therefore, recombinant proteins containing the His-Tag are significantly purified in a single step.

The recombinant protein is generally eluted either with the lowering of pH or with the addition of imidiazole to the column.

Advantages:

• Hexahistidine tag is much smaller and hence provides high yields of tagged proteins.
• It does not interfere with the structure and function of the recombinant protein.
• Its affinity for Ni-NTA matrix is non-dependant on the conformation of the target protein.
• The elution conditions for His-tagged protein are milder.
• Ionic strength, chaotropic agents and detergents do not affect the purification of His-tag proteins.
• Less expensive.

Because of all these advantages, His-Tag is the affinity tag of choice for various protein expression and purification experiments.

The only disadvantage of the tag is that certain bacterial proteins are able to bind to the His-Tag and hence are co-eluted with the recombinant protein. This can be partially ruled out by increasing the stringency of washing.

2)  GST-Tag

The Glutathione S-transferases (GSTs) are class of enzymes that are involved in cellular defense against small toxic compounds. They are abundant enzymes that utilize glutathione as a substrate. GSTs bind to glutathione with high affinity and specificity. Therefore, they provide an efficient system for the purification of proteins.

Generally, the Glutathione S-transferase (GST) from Schistosoma japonicum is used as the affinity tag in the pGEX vector series. The gene encodes a protein of 218 residues having molecular weight of 26KDa. The fusion protein thus produced can be purified using the glutathione-based affinity resins. The strength and selectivity of the resin for GST-tagged proteins results in the successful purification of the recombinant protein from the cell extract, in a single step.

In order to elute the recombinant proteins, reduced glutathione is added to the column. This allows the elution of recombinant proteins under mild and non-denaturing conditions.

Advantages:

• Provides a higher degree of purification in a single chromatographic step.
• Increases the solubility of the recombinant protein.
• It does not interfere with the structure and function of the recombinant protein.
• Provides immunogenic as well as biochemical assay of the recombinant protein.
• The elution conditions for GST-tagged protein is milder than most of the affinity purification methods.

Disadvantages:

• Due to its larger size it is prone to degradation by proteases.
• Affinity for glutathione resin depends on certain reagents.
• More expensive.
• In order to study the protein of interest in detail the tag has to be removed.

Nonetheless, GST-tag is a versatile tag for protein expression and purification. It is generally used when protein of interest is not expressed in His-tag system or is having solubility or purity issues.

3)  MBP-Tag

Maltose Binding Protein (MBP) is a periplasmic protein of the bacterium E.coli. The protein is a component of the maltose and maltodextrins. It is encoded by the gene malE. The malE gene product bears a wide variety of fusions and hence is suitable for expressing proteins which do have problems in expression, in His-Tag or GST-Tag systems.

The expression vector system consisting of MBP as the affinity tag is pMAL series (New England Biolabs).

As MBP is a component of maltose, therefore, the fusion protein can be purified using matrices consisting of sugars. Generally amylose resins are used for the purification of MBP tagged proteins. For the elution of the recombinant protein maltose is added to the column. As a result of this, the elution of recombinant proteins is mild and under non-denaturing conditions.

Advantages:

• Provides a higher degree of purification in a single chromatographic step.
• Increases the expression as well as solubility of the recombinant protein.
• It does not interfere with the structure and function of the recombinant protein.
• Allows periplasmic expression of the recombinant protein.
• Allows the formation of disulfide bonds in the foreign proteins.
• Limits degradation of the recombinant protein.
• Less expensive.

Disadvantages:

• Due to its larger size, expression of large proteins is sometimes problematic .
• In order to study the protein of interest in detail the MBP tag has to be removed

The elution conditions for MBP-tagged protein is milder than most of the affinity purification methods. In addition, due to bacterial origin of malE gene, MBP is a better tag in terms of expression and solubility as compared to GST.

These were the overview of the three most commonly used  affinity tags for the purification of heterologous proteins in E.coli. According to our experiences, one should start with the His-Tag, followed by GST and MBP. In most of the cases, His-Tag results in decent level of expression and purification of recombinant proteins. For any query or suggestions related to protein purification in E.coli, please feel free to post it in the comments.

Kuby Immunology 4th edition by Richard A. Goldsby, Thomas J. Kindt and Barbara A. Osborne

Kuby Immunology is one of the top immunology textbooks on the academic market. Currently in its 6th edition, Kuby Immunology is written by a staff of top instructors in immunology.  The textbook is named after Janis Kuby, a former professor at San Francisco State University and the writer of the original edition.  It was originally produced as a textbook entitled Immunology, but became so well regarded in the field that it became officially designated as Kuby Immunology.  Kuby Immunology is, by and large, designed as an introductory textbook, and is thus used primarily on an undergraduate level in Pre-Med and Biology programs.  Kuby Immunology is highly regarded for being consistently comprehensive and up-to-date with many of the most crucial discoveries in the every changing and developing science of immunology.  Since it is designed as introductory, Kuby Immunology writes out many of its concepts in straight, easy to understand terms, largely avoiding more complicated academic language.  It covers most of the various  categories of immunology, and focuses greatly on the multi-disciplinary nature of immunology since much of its audience does not necessarily pursue careers in immunology.  For them, Kuby Immunology is the main text that they will ever read about immunology. 

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Encyclopedia of Microbiology

Humans have always struggled with how to balance the benefits bacteria offer with the threats that they produce. Much less obvious than the effects microorganisms have on plants and animals are the indirect ways in which they shape the planet. These hidden activities have rarely been explained in science, though scientists realize that the behavior of microbes supports all life on Earth.

In more than 200 entries, Encyclopedia of Microbiology presents the myriad ways in which microorganisms influence the biosphere. Focusing on how all microorganisms relate to each other just as all higher organisms relate to all other animate and inanimate objects on Earth, this new resource explores all aspects of microbiology from mycology (the study of fungi) to the simplest biological entities of all, viruses, to prions, which are even more streamlined than viruses and just as dangerous. Biographical sections in many entries highlight the scientists who most influenced developments or discoveries in microbiology, including Louis Pasteur. Entries cover new techniques in microscopy, genetic engineering, gene therapy, and nanotechnology. A full-color insert, helpful appendixes, cross-references, and further resources round out this invaluable resource.

Essays include:

• Where Are Germs Found?
• The Realities of Biological Weapons
• Does Immigration Lead to Increased Incidence of Disease?
• Will Global Warming Influence Emerging Diseases?
• Microbes Meeting the Need for New Energy Sources
• Bioengineered Microbes in the Environment
• Does Vaccination Endanger or Improve Our Health?
• Does Air Travel Make Us Sick?

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5 Factors affecting gene cloning efficiency

Gene cloning is the central technique involved in recombinant DNA technology. Moreover, it facilitates the discoveries and understandings of the gene structure, function and regulation. A new era has been initiated as a result of this method in the manipulation, analysis and exploitation of bio-molecules.

However, competent gene cloning is not that much easy as it sounds. In order to efficiently clone the gene of interest into a particular vector, you need to be skilled. Since the efficiency of cloning is determined by several factors. Therefore, each factor should be deliberately considered to get best cloning efficiencies.

Here we provide you the most common factors which affect the efficiency of your gene cloning experiment.

1)         Starting Material

Well begun is half done. You know this notion very well. This applies to gene cloning also. A good starting material means you have done half of the things correct, only remaining half is to be optimized.

You materials should be pure. The isolated plasmid should be free from contaminating components. One of the most common contaminant is the media in which the culture is grown. This results in the poor digestion and ligation of the plasmid.

Therefore, during plasmid isolation, you should make sure that the media has been completely removed from the bacterial cells. Moreover, it is also a good practice to ethanol precipitate the plasmids prior to restriction digestion. This removes the salts present in the plasmid suspension and hence results in proper digestion of the plasmids.

If PCR product is the starting material then either reaction cleanup or gel extraction should be performed. We generally go for the gel elution of the PCR product as this not only removes the reaction components and primer dimers but also the non-specific amplifications.

2)         Digestion of the Vector and Insert

Digestion is very important factor determining the efficiency of the cloning experiment. Good cloning efficiency requires complete digestion of the insert and vector molecules. Digestion, if not properly done results in partial digestion of the vector and insert molecules, which in turn results in poor cloning efficiency.

However, it is relatively uncomplicated to achieve complete digestion. You should follow the stuffs quoted here in order to get desired results.

You should take units of the restriction enzyme according to the amount of plasmid or PCR fragment taken. Generally, 1-2µg of plasmid DNA/PCR product is a good amount for digestion. To digest this amount of DNA you should take 1-2µl (5-20 units) of the chosen restriction enzyme. Now-a-days many suppliers provide enzymes in a format that contains units in 1µl to digest 1µg of DNA. This really simplifies the digestion process.

Things become more complicated when you have to perform double digestion. While performing double digestion you should always check the buffer in which both the enzymes are having maximum activity. But there are certain enzymes which cannot be used for double digestion. The reason being the fact that there buffer requirements are not compatible. In that case it is better to perform sequential digestion.

However, sequential digestion results in the loss of the fragment so you will have to start with more amount of DNA. In addition, there are certain enzymes which have different optimum temperatures. In this case also you will have to go for sequential digestion.

However, there are certain suppliers (NEB, Fermentas etc) who have optimized a single buffer and a single temperature for a variety of commonly used enzymes. Unfortunately, these enzymes are little bit expensive but in our opinion are worth. Using such enzymes not only saves your time and energy but also improves the digestion many folds.

3)         Amount of digested vector to be taken for ligation

Well, after completely digesting the insert and the vector molecules, the question arises about the quantity of digested vector to be used for ligation. This significantly affects the efficiency of your gene cloning experiment.

The amount of vector to be taken solely depends on the size of the vector. For example, if the size of the vector is small then you will have to take little amount of vector and vice-versa.

We have made a generalized approach towards the size and amount of vector to be taken. According to our experiences, the optimum relation between size and amount of digested vector is as follows:

Size                                        Amount

<5 kb                                      50 ng

5-7.5 kb                                  75 ng

7.5-10 kb                                100 ng

> 10 kb                                   upto 150 ng

Taking amount of digested vector according to its size increases the ligation to a great extent and hence the cloning efficiency.

3)         Insert vs Vector Molar Ratio

Molar ratio plays a valuable role in the ligation of the fragments and hence in the cloning efficiency. But before moving further, let us see what molar ratio is? Molar ratio (sometimes also called as molar excess) is the amount of moles of insert per moles of vector molecule.

Generally, molar ratio is taken 3. That is, three insert fragments per vector molecule. It is a good practice to calculate the amount of insert in respect of vector and molar ratio prior to setting up the ligation reaction. The formula is:

ng of insert =  ^{amount of vector x molar ratio x size of insert} /size of the vector

For example, if the size of your vector is 6 kb and the size of insert is 1200 bp, then for setting up the ligation reaction you should take 75 ng of vector and 45 ng of insert.

However, if the difference between the size of insert and size of vector is more (e.g. insert=300 bp; vector=12000 bp), then molar ratio should be 5-10.

In order to accurately quantify the amount of eluted insert and vector molecules you should either use spectrophotometer or a nano-drop. However, we will not recommend you to estimate the amounts by running the samples in agarose gel. This does not allow accurate quantification of the fragment and hence results in lower ligation and cloning efficiency.

5)         Efficiency of the competent Cells

All well that ends well. This is also the case with gene cloning. If you have done all the things correct but you are not doing the last step properly, then you will ultimately ruin your whole hard work.

The ligation mix is used to transform E. coli competent cells and hence efficiency of the competent cells used significantly affects the cloning efficiency. We will recommend you to use high efficiency competent cells for your all cloning experiments. Always use the protocols which results in transformation efficiency of > 10⁷ cfu, for preparing competent cells.

However, if you are unable to prepare high efficiency competent cells, we will recommend you to go for commercially available competent cells. Nevertheless, you can easily prepare high efficiency competent cells, by going through our forthcoming article on competent cell preparation.

We hope that these informations will be helpful for your gene cloning experiments. However, if you have any further query regarding gene cloning, please feel free to post it in the comments.

cDNA Synthesis: Principle and Procedure

With the advancement in the field of genetic engineering, gene expression analysis has become an indispensable tool. Researchers are always keen to find out whether their gene of interest is expressing (turned on) or not (turned off). For this, the mRNA (messenger RNA) is located and quantified in the given sample. mRNAs carry the information coded by DNA and, thus, further gets translated to produce respective proteins.

RNAs are very unstable and fragile, and are very likely to degrade by the omnipresent RNases. In order to combat this, the biological informations encoded in mRNA are stored in more stable form of nucleic acid, i.e. DNA. Therefore, cDNA is prepared from RNA, which stores entire sequence of the mRNA. It is more convenient to work with cDNA as compared to mRNA. This cDNA can be further used for various subsequent molecular biology and genetic studies.

What is cDNA??

cDNA means complementary DNA or copy DNA. According to the central dogma of the molecular biology, DNA is transcribed into mRNA. Then mRNA gets translated to produce protein. Therefore, the flow of biological information is from DNA to RNA to protein.

However, sometimes the flow of information is from RNA to DNA (as in the case of some viruses, e.g. HIV). This conversion of RNA to DNA is aided by an enzyme known as Reverse Transcriptase (i.e. RNA-dependent DNA polymerase). The cDNA prepared can be single stranded or double stranded. Therefore, molecular biologists make use of reverse transcriptase to prepare cDNA from mRNA for the sake of convenience in the molecular studies.

Principle of cDNA synthesis

Mature (fully spliced) mRNA is used as a template for preparing cDNA. In fact, cDNA can be produced from any RNA molecule. This conversion is brought about by reverse transcriptase. cDNA can be obtained both from prokaryotes and eukaryotes.

Reverse transcriptase is a RNA-dependent DNA polymerase. It acts on a single strand of mRNA. Using mRNA as a template, reverse transcriptase produces its complementary DNA based on the pairing of RNA base pairs. This enzyme executes reactions in the same way as DNA polymerase. It also requires a primer with a free 3′-hydroxyl group.  For transcribing RNA having secondary structures, a reverse transcriptase with high temperature performance is recommended.

Procedure of cDNA synthesis

First of all, good quality intact mRNA or total RNA is isolated. Then, you need a few more reagents to prepare cDNA: dNTPs (dATP, dTTP, dCTP, dGTP), primers and reverse transcriptase.

In case of eukaryotic mRNAs, a poly-A tail is present at their 3′-ends. Therefore, a poly-T oligonucleotide is used as a primer. But certain modifications are needed when you use other RNAs which lack poly-A tail, e.g. prokaryotic mRNA, rRNA, RNA virus genomes, etc. In such cases, a poly-A tail is added to the 3′-end of the RNA. This makes it analogous to the eukaryotic mRNA.

The primer gets annealed to the 3′-end of the mRNA. Now, the 3′-end of the primer is extended with the help of the reverse transcriptase using mRNA strand as a template. This is known as “first strand reaction”. As a result of this, RNA-DNA hybrid molecule is produced. By the use of RNase H or alkaline hydrolysis, the RNA strand of this RNA-DNA hybrid molecule is digested. Now, the single stranded cDNA becomes free.

The reverse transcriptase used (most commonly used is Moloney Murine Leukemia Virus Reverse Transcriptase, MMLV RT) displays terminal transferase activity on reaching the end of the RNA template. It adds 3-5 residues (usually dC) to the 3′-terminal of the first strand cDNA. An oligo containing a stretch of G residues is used. This oligo gets annealed to the dC rich cDNA tail and serves as an extended template for reverse transcriptase. Now, the synthesis of the complementary strand of the first strand cDNA begins. This is called “second strand reaction”. Finally, a regular double stranded DNA is produced.

Types of primers used

Various types of primers can be used, in accordance to the requirements, to synthesize cDNA.

1) Oligo-dT primer- It is used when the mRNAs have poly-A tail, as in the case of eukaryotic mRNAs; or when a poly-A tail is attached to the existing RNA. Oligo-dT primer anneals to all the mRNAs simultaneously.

2) Sequence-specific primer- If you wish to generate cDNA from a particular population of mRNA among all the mRNAs, then sequence-specific primer is used. It will bind to a particular mRNA sequence only. This will give rise to a pure cDNA population generated from the desired mRNA. For designing sequence-specific primer, you must know the sequence of the mRNA of interest. Generally, the 3′-terminal sequence is preferred.

3) Random primer- A random primer cocktail is used to produce cDNA from all the mRNAs. The cDNAs produced are not full length. Random primer is extremely useful if production of the shorter cDNA fragments is desirable. Its use increases the probability of converting the entire 5′-end of the mRNA into the cDNA. In case of long mRNAs, reverse transcriptase is usually not able to reach the 5′-end. Therefore, random primer proves to be extremely advantageous in such cases.

Types of cDNA

cDNAs can be single stranded or double stranded. After the first strand reaction, cDNA obtained is single stranded. This single stranded cDNA can be converted to the double stranded form by second strand reaction. On the basis of the applications, single or double stranded form of the cDNA is used.

Applications of single stranded cDNA

1)      Single stranded cDNA is most commonly used for RT-PCR (Reverse Transcriptase-Polymerase Chain Reaction). RT-PCR is done for gene expression studies. It determines whether the gene of interest is expressed or not, and the level of its expression.

2)      It is also used to amplify particular gene of interest. For this, sequence-specific primers are used.

3)      Real-time PCR (also known as quantitative RT-PCR, qRT-PCR) also makes use of single stranded cDNA. It is done for performing gene expression analysis. As the amplification progresses, the amplicons can be visualized with the help of a fluorescent reporter molecular. It is highly sensitive and effective as compared to RT-PCR.

Applications of double stranded cDNA

1)      Double stranded cDNAs are used to clone them into the appropriate vector to prepare libraries of cDNA (i.e. cDNA libraries). These libraries contain all the mRNA sequences in the form of cDNA, which are all expressed in a cell.

2)      Double stranded form of a particular cDNA of interest can be cloned. Then, expression of the desired genes is allowed at the RNA and protein level for further study.

3)      Sequencing of the double stranded cDNA is carried out to obtain the expressed sequence tags (ESTs).

4)      They are also used for doing microarray for analysing global gene expression.

5)      Suppression subtractive hybridization (SSH) is also performed with double stranded cDNA. It is done to find out differential gene expression.

Lyophilization

Biological materials often are dried to stabilize them for storage or distribution. Lyophilization also called freeze-drying, is one method of drying biological materials that minimizes damage to its internal structure

Competitive ELISA

In competitive ELISA, unlabeled antibody is incubated in the presence of its antigen. Then these bound antibody/antigen complexes are then added to an antigen coated well. After washing, unbound antibodies are removed. The more analytes in the sample, the less antibodies will be able to bind to antigens in the well. The signal is then detected using labeled secondary antibodies and the decrease in signal is compared to a control. The major advantage of a competitive ELISA is the ability to use crude or impure samples and still selectively bind any antigen that may be present.

Indirect ELISA

The indirect ELISA is used primarily to determine the strength and/or amount of antibody response in a sample. In the assay, the antigen of interested is immobilized by direct adsorption to the assay plate. Detection of the antigen can then be performed by using a matched set of primary antibody and conjugated secondary antibodies.

Sandwich ELISA

The Sandwich ELISA measures the amount of analyte between capture antibody and detection antibody. The analyte needs to have two different epitope sites available for antibody binding.

Ampicillin

Ampicillin is one of the most widely used antibiotic in molecular cloning, for the selection of transformants. It belongs to the penicillin group of antibiotics i.e. beta-lactam antibiotics. The only difference between penicillin and ampicillin is the presence of amino group in the latter.

Unlike penicillin which is effective against only gram positive bacteria, ampicillin is also effective against gram negative bacteria, e.g. E. coli. The amino group present in the side chain of the ampicillin renders it to penetrate the outer membrane of gram negative bacteria and hence enter into it. Therefore, it was one of the first broad spectrum penicillins to be used.

Mode of action

Like other beta-lactam antibiotics, ampicillin also inhibits cell wall synthesis in the bacteria. It basically inhibits the formation of glycan moiety of the peptidoglycan layer of the batcerial cell wall. The peptidoglycan is known to provide the rigidity to the bacterial cell wall.

Ampicillin is basically a competitive inhibitor of the peptidoglycan synthesizing enzyme transpeptidase. Inhibition of the enzyme, ultimately results in the lysis of the bacterial cells.

Ampicillin resistance gene

The gene responsible for conferring ampicillin resistance is called bla gene and codes for a TEM1 ?-lactamase enzyme.  The TEM1 ?-lactamase hydrolyzes the ?-lactam ring of the ampicillin. This results in the inactivation of the antibiotic. The enzyme is usually secreted into the periplasmic space where it catalyzes the hydrolysis reaction.

TEM1 ?-lactamase is the major ?-lactamase responsible for ampicillin resistance in various gram negative bacteria including E. coli. As a result, this gene is widely used to confer resistance against ampicillin. There are a variety of vectors using bla gene to confer resistance against ampicillin for the selection of transformed E. coli.

Preparing the ampicillin stock solution

The stock solution of ampicillin is prepared in water. Generally, ampicillin sodium salt is used for its better solubility. The concentration of stock solution is generally in the range of 50-100mg/ml. We usually prepare 100mg/ml stock solutions.

Weigh 100mg of ampicillin sodium salt, dissolve it in 1ml MQ grade water and filter sterilize it by using 0.22µm filter. Store the stock at -20?C.

You should note that pH of the water has great impact on the stability of the antibiotic. It is better to use pH 6.8-7.0, of water for preparing the stock. Moreover the stock can be stored for one month only. After that the antibiotic starts to degrade.

Working concentration of ampicillin in media

For broth or agar plates the concentration of ampicillin is kept between 50-100µg/ml.

Problem of satellite colonies

As the beta lactamase is secreted in the periplasm, it degrades the ampicillin nearby the transformed cells. This results in the growth of small untransformed colonies in close proximity of the transformed one. These colonies are called as satellite colonies.

One way to deal with satellite colonies is to incubate the plates for less than 14 hours. Another way is to increase the concentration of ampicillin in the plates. We generally use 100µg/ml of ampicillin in the plates. But here also it is better to incubate the plates for less than 16 hours.