4 replies to this topic

[Bpaul]

Hey All

I am trying to express a GST-tagged protein (gene cloned in pGEX-4T-2 vector!). My protein is around 24 kDa. I did check the reading frame and all that and its all good! I tried expressing the protein in BL21(DE3) at different temperatures (37, 30, 25 and 16 degC) with different IPTG concentrations (0.1 mM and 1mM). And now i see proper bands around 25 kDa (checked using SDS-PAGE and western blots!) and i believe its the GST! Whereas my required protein (25 kDa + GST) is no where to be found!

I am thinking either the protein is not expressing or its expressing but the sonication must be making the GST to fall off?? I am yet to check the uninduced one! Any suggestions? Any ideas? Thank you very much!

BP

[ascacioc]

Your protein is not soluble in the conditions you are trying. When a GST tagged protein leads to the 25 kDa band and not full protein, that means that the cell does not like the protein (either toxic or insoluble, or not properly folded) and it is cleaving it. GST is not a tag that improves folding. It is a tag tag gives false solubility. Better use a MBP tag or a NusA tag. Here a paper talking about this:
http://www.ncbi.nlm....pubmed/16168669 (let me know on private if you cannot access the article, I can send it to you)

You have already tried different expression conditions in this vector. People usually start with 3 different tags in 3 different vectors with different promoters of different strengths (hence they clone in parallel 9 constructs) to try them:
http://www.ncbi.nlm....pubmed/20814932
http://www.ncbi.nlm....pubmed/18678258 (this one has a nice scheme on the second page about what I mean above)
You can also try chaperones system (co-expressing your protein with different chaperones)
http://www.takarabio.../pdf/hd/BV1.pdf
Or in ArcticExpress:
http://www.genomics....tail&PageID=467
since chaperones do not function at low temperatures, in these cells people have cloned some chaperones from sychrophilic bacterium Oleispira antarctica. This makes it possible to both use chaperones and low temperatures.
Another thing you can try with the construct you have now is the autoinduction media, sometimes in this the protein is folded better:
http://www.ncbi.nlm....pubmed/15915565

There are many things you can try. There is no direct way than trying. The order is up to you. Good luck,

Andreea

[Bpaul]

Thank you very very much!!!!!! Will try it!!!! and will let you know! Thanks again! :-)

[Bpaul]

ascacioc, on 29 August 2012 - 10:34 PM, said:

Your protein is not soluble in the conditions you are trying. When a GST tagged protein leads to the 25 kDa band and not full protein, that means that the cell does not like the protein (either toxic or insoluble, or not properly folded) and it is cleaving it. GST is not a tag that improves folding. It is a tag tag gives false solubility. Better use a MBP tag or a NusA tag. Here a paper talking about this:
http://www.ncbi.nlm....pubmed/16168669 (let me know on private if you cannot access the article, I can send it to you)

You have already tried different expression conditions in this vector. People usually start with 3 different tags in 3 different vectors with different promoters of different strengths (hence they clone in parallel 9 constructs) to try them:
http://www.ncbi.nlm....pubmed/20814932
http://www.ncbi.nlm....pubmed/18678258 (this one has a nice scheme on the second page about what I mean above)
You can also try chaperones system (co-expressing your protein with different chaperones)
http://www.takarabio.../pdf/hd/BV1.pdf
Or in ArcticExpress:
http://www.genomics....tail&PageID=467
since chaperones do not function at low temperatures, in these cells people have cloned some chaperones from sychrophilic bacterium Oleispira antarctica. This makes it possible to both use chaperones and low temperatures.
Another thing you can try with the construct you have now is the autoinduction media, sometimes in this the protein is folded better:
http://www.ncbi.nlm....pubmed/15915565

There are many things you can try. There is no direct way than trying. The order is up to you. Good luck,

Andreea

Hi

I checked the uninduced supernatant and found we have leaky expression! So i used 1% glucose to reduce the leaky expression! Leaky expression has gone but also no expression even after IPTG induction! Could it be because am using too much glucose> Or do you think i should just ditch this construct and try something else?

[ascacioc]

I am not familiar with this glucose adding protocol. I have heard about it and I theoretically know how it functions. But my impression is that you cannot control when the glucose is used up: different bacteria with different plasmids and different protein constructs will grow differently and they will use the glucose differently. So, I think that you just have to wait a bit longer with the induction. Or check later time points for induction. Because the IPTG will be ignored by bacteria as long as they have glucose. I would recommend the auto-induction media for your purposes. Alternatively, I would change the expression strain to smth that has the LacI/LacIq gene in the chromosome or on another plasmid, additionally, to make it even tighter (Lucigen or NEB had these strains with extra LacI) Or...change the construct to a tighter promoter, such as BAD.