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Thursday, 9 June 2011

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

11 comments:

  1. keep writing! i just love ur blog.really helps!!

    ReplyDelete
  2. Hi Maciek,
    Found this blog post really helpful! I couldn't understand the protocol I was given for this! . . . Just wondering if you could share with us which textbook you got your images from please?

    ReplyDelete
  3. Hi Maciek,
    I found this blogpost really helpful so thanks for putting it up! I couldn't understand the protocol I was given for this experiment but I found this really helped. . . I was just wondering if you could please share with us the name of the textbook which you got the images from?

    ReplyDelete
  4. Dear Rachel and She's a lady,

    Unfortunately, these pictures were made by me and they do not come from any textbook. However, if you need help with understanding something else please let me know and I will try to help you.

    Cheers,

    Maciek

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  5. Hi Maciek,
    I found this blog really helpful and diagrams are great. Just wondering where the protocol comes from?
    Thanks
    Sinéad

    ReplyDelete
  6. Dear Sinead,

    I made this pictures myself. If you need them please download and use as you wish.

    Let me know if you need any other cartoon I can make it for you.

    Cheers,

    Maciek

    ReplyDelete
  7. Hi! This post is really... great! ^_^
    I just have one question: do you know if ther's a scientific article where I can find some of the information you talk about?! For example I would like to find a picture of a gel like the one you posted...
    Thanks for all... due to the fact you're reading this comment... or rather "translating" this comment! I'm really sorry for my english! :)

    ReplyDelete
  8. Hi,

    www.ncbi.nih.gov - search in pubmed.

    Cheers,

    Maciek

    ReplyDelete
  9. Hi Maciek,

    Nice protocol! Well, I have a question here that how did you usually remove the GST from your targeting fusion protein?

    Many thanks,
    Hao

    ReplyDelete
  10. Dear Hao,

    this is usually achieved by digestion with a specific protease. The GST vectors (pGEX vectors that I usually use) have aminoacid sequences between GST and your target protein that are recognized by different proteases such as Thrombin, Factor X or others. After the purification of the GST-fusion, you incubate your elutions with the appropriate protease (according to manufactuter suggestions) and later capture the GST moitety with the GSH beads. However, if you gonna do this I advise you to do few small scale digestions and check for the solubility of your GST-free target protein. Please remember that GST very often helps to keep the target protein in the solution and when they separate some proteins tend to precipitate (especially on GSH beads that you later use to remove free GST). If this is the case, then it may be better for you to clone your target protein in to non tagged bacterial plasmid and express it without any tag. Most of the bacterial proteins have low pI whereas human proteins for example have relatively high pI. This allows for the purification of the target protein using the ion exchange chromatography + some other polishing step such as heparin column if your protein is DNA/RNA binding protein. Believe me I tried that and it works much better than GST purification (this may be another post:). I hope it helped but if you have more questions, do not hesitate to contact me again on maciejk@sund.ku.dk

    Cheers,

    Maciek

    ReplyDelete
  11. Excellent piece.For the latest on biochemistry including research visit BiochemistryBlog.com

    ReplyDelete