GGS LIVE - Site Directed Mutagenesis

Hello BioFreakers!!

Today in the GGS - LIVE section the Site Directed Mutagenesis technique. Wanna see how it is done? Lets roll then.

Method: Site Directed Mutagenesis (SDM).

About: Allows for introduction of point mutations in DNA and thus sometimes in protein sequence.

What: Introduction of STOP codon in order to generate C-terminal deletion in protein X.

         Site directed mutagenesis is performed on circular DNA substrate. In our study case, the cDNA of protein X will be inserted into GST vector. Cloning of the protein X was performed analogously to study case described in a different post (click here GGS LIVE - Makinga fusion protein).

         To mutate protein X cDNA sequence we will need a set of primers that will be used in PCR reaction (GGS LIVE - Polymerase chain reaction (PCR)). SDM primers overlap with the target sequence, whereas regular PCR primers flank the target sequence (see cartoon below).

          The idea behind SDM is that each primer will mutate a single DNA strand of the pPLAS + X cDNA plasmid giving a product of the mutated pPLAS + X cDNA. Naturally, the template will be also present in the final mixture. Tamplate is removed by DpnI, a restriction endonuclease that digest modified DNA (DNA methylation). Such modification of the DNA occurs within bacterial cells, therefore SDM product will not wear it, as it was formed in vitro. After PCR reaction and overnight digestion with DpnI, SDM  reaction is analysed by agarose gel electrophoresis (see figure below).

         As you can see from the gel electroporesis analysis, desired product is present in both DpnI treated and untreated samples (R and R+D). Template +/- DpnI was used as a negative control. Additionally, amplification of 0.3kb fragment served as template control.
         You probably, wonder why SDM product and template are not observed as single band. This is due to different conformations of these plasmids. Template is supercoiled, therefore more packed and travels through gel faster and thus appears smaller on the gel. SDM product is not and therefore migrates slower.

         The R+D sample is then used to transform bacterial cells. In this process specific E.coli cells (for example Top10) uptake R+D plasmid and replicate it. This allows obtaining workable amounts of DNA, which is then send for sequencing. Several different E.coli clones are tested and when sequencing confirms mutation of pPLAS + X cDNA, DNA from correct clone is used to transform yet another specifc E.coli strain (for example BL21 pLysS).
          We then express X and X-truncation proteins using these BL21 cells (see figure below). For the protein expression in E.coli tutorial, please go here GGS LIVE - Protein expression in E.coli.

As you can see, this way we can generate a mutant protein KA-CHING. 

I hope you enjoyed it.

Maciek

GGSTEAM

GGS LIVE - Protein purification from E.coli

Yo Yo Biofreakers,

Today we are going to have closer look at the recombinant protein purification from bacterial cells.

Method: Purification of recombinant protein.

About: Having an optimised protocol for protein  purification is an essential tool to study properties of the protein of interest.

What: Purification of GST-tagged chicken protein previously expressed in E.coli.

Before we start we should first have a look at what the GST tag is. GST (glutathione S-transferase) is an enzyme that transfer glutathione (GSH) via slufhydryl group to differernt type of substrates (lipids, xenobiotics). GST has a high affinity towards its substrate glutathione and this property of GST is utilised to purify GST-tagged proteins. In order to recover GST-tagged protein from the complicated mixture of proteins, the fusion protein is incubated with agarose beads coupled to glutathione (GSH-agarose, see picture below), what leads to efficient precipitation of GST-fusion protein.

Ok Vamos!! Expression of the GST-X protein was previously demonstrated in different post GGS LIVE - Protein Expression in E.coli.


After a succesfull expression of the protein of interest in E.coli, we can purify it using one of the many protocols available for GST protein fusion purification. In general such protocol consists of three major steps: cell lysis and solubilisation of the GST-fusion protein (see cartoon below), recovery of the fusion from the lysate and elution of the GST fusion.

As you can see on the cartoon above, first GST-X fusion is expressed in large amount (for example 250 ml to 1 l culture) under previously optimised conditions (for protein expression see post GGS LIVE - Protein expression in E.coli). After protein expression, E.coli cells are harvested by centrifugation, superatant is removed and cell pellet is resuspended in lysis buffer of choice. Usually such buffer should have pH of around 7.0 - 8.0 to facilitate efficient interaction between GST and its substrate glutathione, protease inhibitors (such as PMSF) to prevent protein degradation. Additionally, lysis buffer should contain component taht will help lyse the cells, such as lysosyme (enzyme that degrades bacterial cell wall) or detergent (which disrupts bacterial cell wall). Cells are usually lysed at 4C rocking or mildly shaking what increases lysis efficiency. Cell lysate is then sonicated to share bacterial DNA (DNA makes lysate viscous and hard to work with) and help to solubilise proteins by breaking up protein aggregates. In the next step, cell debri is removed by high speed centrifugation. And there we go we have a lysate ready for protein purification.

As mentioned earlier, in order to recover our GST-X protein we have to mix our lysate containg the fusion with glutathione agarose beads. First beads have to be prepared (see cartoon below).

Glutathione agarose beads are first washed with the lysis buffer in order to remove storage solution (usually ethanol, which can impede binding of GST to glutathione). Then beads are mixed with lysate containing GST-X fusion and incubated at 4C in order to bind GST fusion to the beads. After binding step, beads have to be washed in order to remove unbound GST-X fusion and unspecifically bound proteins.

At this stage of purification GST-X fusion should be clean and depending on the nature of furhter experiments that we want to perform, we can either elute the fusion with glutathione (excess of the glutathione will compete and displace GST-X protein from the beads) or cleave the GST tag and release protein X (see cartoon below).

When purification is finished  we can analyse our experiment by separating protein sample taken at each step of the purification by SDS-PAGE and stain proteins in gel with Coomasie dye. The results from GST-X purification are shown on the picture below.

As you can see from the Coomasie stained gel, the expression of GST-X fusion was nicely induced (UI and I samples). We can also confirm that the GST-X was present in the starting material (lysate IN sample). After incubation of the lysate with beads, most of the GST-X bound to the resin what resulted in depletion of GST-X, as observed in unbound sample (UN). After beads wash, a single band of GST-X was detected on the beads, indicating high purifty of this sample. Elution of the GST-X with glutathione recovered fusion protein from beads. Additionally, alternative elution by GST cleavage resulted in appeareance of two bands: a free X protein and GST tag.

Hopefully, you got the picture how protein purification can be performed using a GST tag as a bait.

I hope u enjoyed it.

Cu SOON!

Maciek

GGSTEAM

GGS LIVE - Protein expression in E.coli

Dear BioFreaker,

today in the GGS LIVE section we are going to look at the recombinant protein expression in E.coli.

Method: Protein expression.

About: Protein expression in E.coli is a widely applied technique, which allows for quick and robust production of the particular protein. Such protein can be then used in different applications, such as antibody production, in vitro enzymatic and binding assays, crystallisation etc.

What: Expression of the GST-tagged chicken protein in E.coli.

          Ok, lets start. If we want to express any protein in the bacteria we have to first clone it (for more info about cloning please visit this post GGS LIVE cloning) into a plasmid which contains specific elements allowing for protein production within the prokaryotic bacteria cells. These plasmids utilise technology based on application of lac operon system (click here for more information about the lac operon system). We will not cover how this system works in this post, we will just go straight to the results:). The only thing you should know at this stage is that protein expression from the lac operon system is achieved by addition of IPTG compound. The IPTG turns on the protein production, which cannot happen without it.
            If a technology for protein X purification is established then expression of such can be performed using native protein sequence. If such protocol is not available we can express our protein as a fusion. The tag which will be added to the protein will later allow for its rapid and robust purification. There are many different tags available for various applications, such as His, GST, MBP, CBP, S-tag, FLAG, Strep and many more. In our study case we will work with GST-tagged protein. If you would like to know how to tag a protein of interest, please look at this post GGS LIVE - Making a fusion protein.
          After we cloned our DNA sequence of interest into a N-terminal GST tag containing vector, we have to transform E.coli strains with this plasmid and then find optimal conditions for expression of our fusion protein. This step has to be performed experimentally. The culture of bacterial cells is split into many batches where protein expression is performed at different conditions, such as IPTG concentration, temperature, time, different media composition etc. At the end of the experiment samples are separated by gel electrophopresis and proteins visualised by staining of the gel. Such gel staining, where protein expression at different temperatures was tested, is shown on the picture below:

Picture taken by Kliszczak M.

          I have to mention that our protein of interest has size of 27 kDa and the GST tag is similarly big  (28 kDa), what gives size of 55 kDa for the fusion. As you can observe on the gel above, increase of temperature during expression positively affects production of GST-X protein by E.coli cells. The 37C seem to be the best temperature for expression of fusion protein. In addition you can observe that GST-X has the predicted size of 55 kDa. After, establishment of perfect conditions, these can be used for production of our protein.

I hope you enjoyed. In the next post we will cover the purification of the GST fusion protein from E.coli.

Maciek GGS TEAM

GGS LIVE - Polymerase chain reaction (PCR)

Today Biofreakers PCR!!

Method: Polymerase chain reaction (PCR).

About: PCR allows for amplification of a particular DNA sequence.

What: Ampliifcation of a gene X sequence from genomic DNA.

Ok lets start. Polymerase chain reaction is a commonly used technique. The theory behind it as well aas practical part of PCR are very simple. For PCR reaction we need:
- thermostabile polymerase - which can stand temperature up to 95 degrees Celsius,
- primers - short DNA oligonucleotides that determine DNA sequence to be amplified,
- DNA template - contains target DNA,
- buffer - supply perfect conditions for polymerase activity.

As I mentioned before theory of PCR reaction is very simple. Cartoon below represents major steps in PCR protocol:

The steps 2 - 4 are repeated between 15 - 30 times in order to obtain a workable amount of the DNA product. Time of the PCR experiment depends on the product size. After the PCR reaction small amount of the reaction mix (approximatelly 5-10%) is analysed by the agarose gel electrophoresis. Example of the gel is shown on the picture below:

As you can observe we did amplify two different DNA sequences. The positive control reaction was set up with primers that previously amplified the desired sequence from the same DNA template (product size 2.5 kb). As expected there is no signal in the negative control lane as negative control reaction did not contain the DNA polymerase. The band in the P lane (the experiment PCR reaction) need to have an expected size. Our gene of interest is 3.9 kb long.

As you can see this way we can very easily ampllify a DNA sequence of interest in order to study its sequence or cellular function.

I hope you enjoyed.

Maciek

GGSTEAM

GGS LIVE - Foci kinetics

Yo, Yo, Yo Biofreak readers!!

Method: Foci kinetics.

About: Foci kinetics assay is performed in order to assess foci formation and resolution of particular protein under specific conditions.

What: Kinetics of gamma-H2AX foci formation after ionizing radiation in wild-type and mutant cells.

In our study case experiment we will investigate gamma-H2AX (histone modification) foci formation and resolution after treatment with ionizing radiation. Ionizing radiation cause DNA double strand breaks.
In response to such DNA damage H2AX histone is phosphorylated (called gamma-H2AX in phosphorylated state) to facilitate double strand break repair.
Briefly, DT40 cells were treated with IR (5 Gy), harvested and analysed by immunoflurescence at different times post-IR treatment (for immunofluorescence tutorial visit GGS LIVE - immunofluorescence). Pictures below represent cells stained for gamma-H2AX harvested at different times post-IR treatment.

As you see on the images above there is not much of signal from gamma-H2AX in untreated cells (there is small number of spontaneous DNA damage in unchallanged cells - red arrow heads indicate representative gamma-H2AX foci). After induction of double strand breaks with IR treatment H2AX histone is robustly phosphorylated (15 min timepoint) and this modification is removed with ongoing DNA repair. At this stage we have to quantify our results. We can eitehr simply score number of the foci per cell or score number of cells with more than X foci of gamma-H2AX. Plot below represents such quantification in which cells with 6 or more gamma-H2AX foci were scored as positive.

From the quantification plot you can see that both cell lines induce gamma-H2AX foci fomration with the same kinetics, indicating that this process is not affected in the mutant cell line. When we look at gamma-H2AX foci resolution, four hours post-IR treatment 50% of wild-type cells has resolved gamm-H2AX foci, where mutant cells need another 4 hours to accomplish the same task. Such results indicate that mutant cell line might have problems with repairing DNA damage caused by IR treatment.

I hope you enjoyed:)

Maciek

GGS TEAM