We know that GST has a large protein size, 225.5 KDa. When other tags are available, say 6X His tag, then why is a tag with such a huge molecular weight used which might interfere with downstream applications, e.g., Purification of the fused protein?
Hi there,
GST is 26kD in size (not 255!) which is comparible with other protein domains used as tags. The main interest of these "big" tags is that further to the affinity they have for small molecules they also have a solubilizing/stabilizing effect on the protein of interest. So working with a GST fusion is sometimes the best option to study the protein of interest.
When it comes to protein purification there is always the trade-off between protein yield and protein purity. The His-tag is firmly on the side of protein yield.
The GST-tag fits a good balance because it exploits enzyme-substrate specificity ensureing high purity, but at the same time it has more protein yield than other tags like FLAG than aim for high purity instead of yield.
As you mention the downside is the total size of the tag with 26kDA and the downstream applications. However there is a way around this. Instead of using L-Glutathione for the proteins cleave the tag of off a protease, whose cleavage site you add between protein of interest and Tag.
Here is an example on how this could work:
https://cube-biotech.com/gst-tag-how-a-protein-helps-to-purify-a-protein#removal
Hi there, GST is just 26 kDa tag which has an affinity to get precipitated with glutathione. Therefore, it is easier to work with GST-tagged protein such as in Immunoprecipitation studies. Also, it is economic and can be expressed in bacteria. Hope it helps
Good luck!
Dear Shilpi
the use of N-termial fusion patner (as GST, but also MBP, Trx, GB1, Ztag) can be in some cases (especially for small protein or mammalian protein) usefull to improve the expression level and solubility of the proteins.
GST, similarly to MPB has also the additional properties to be able to bind the glutatione sepharose resin and therefore can be also used for affinity purification of the protein complex or of cleaved GST after incubation with protease.
In my experience there is not a best tag, in the past i was able to obtain best results with GST (eg human sco1 ans sco2) as well as with MBP (mia40) or GB1 in other cases.
im my opinion,. jusy the his tag (removable with TEV cleavage) is in the most cases the choiche, in case it fail, you can try the different N-term tag.
If you are interested to see some very basic experimental data in the following presentation
https://www.blogger.com/video.g?token=AD6v5dzPeA9ws3hcPvrO8fBABN8iFQ0dwI7GIHys9p8dwVtov4dGrKXH9PF91d9bVMYcLxTYY3XJesKt62Y-LsZcaHDCJKZbNW-DLUm_4p5-75Dbj1VZlKTlyyl2aQzf-EPwO3Ac6eBG
available on my blog: PRoteoCool
at minute 8, you can see an SDS page with the expression of GFP tagged with several different tag at the N.term.
good luck
ciao
Manuele
The GST tag can improve the solubility of the protein, as is well the MBP tag.
This video has a full answer to all question you have about GST fusion protein system.
https://lnkd.in/eEANJVni
- Badges
- Science topic
- Similar topics
- Biological Science
- Biochemistry
- Enzymology
- Enzymes