Monday, 14 February 2011

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.