Hello,
I have cloned my gene of interest in a bacterial expression system i.e. BL 21 Gold (DE3) and checked for protein production and solubility. Good amount of protein was observed on SDS-PAGE in the insoluble fraction. I have tried changing the induction temperature from 37°C to 18°C, changing the expression strain, and changing the vector system to include a tag aiding in solubility. Can someone suggest an answer as to how I can bring the protein in the soluble fraction?
Hi, there are many ways and methods how to solubilize your protein. I do not know the size of your final protein or the amount you need to get. But here are some recommendations you can try:
- Use of MBP-tag helps to boost the expression and also to solubilize the final protein. But cleavage needed (thrombin or TEV are working the best)
- Use of His-Tag and express the protein as insoluble, maybe in inclusion bodies. (not bad thing at all) Then resolubilize in 8M urea and do the His-chromatography in 8M urea. You can also use GdnCl (stronger than urea) but you can not do the SDS-PAGE afterwards. After the chromatography you can try to dialyze the sample (not simple at all, but there is a chance).
- In one of my projects, I used MBP at N terminus and His at C terminus, than you can combine the properties of both :-).
- If you do not want to design a new vector, you can also try to reduce the conc. of IPTG, I also used 16°C for some protein and it helped. But prolong the cultivation after induction to 18h min.
- anyway there is much more you can try, just write some more info about your protein. Size, AA sequence, origin, PI,..
BR
Hi, there are many ways and methods how to solubilize your protein. I do not know the size of your final protein or the amount you need to get. But here are some recommendations you can try:
- Use of MBP-tag helps to boost the expression and also to solubilize the final protein. But cleavage needed (thrombin or TEV are working the best)
- Use of His-Tag and express the protein as insoluble, maybe in inclusion bodies. (not bad thing at all) Then resolubilize in 8M urea and do the His-chromatography in 8M urea. You can also use GdnCl (stronger than urea) but you can not do the SDS-PAGE afterwards. After the chromatography you can try to dialyze the sample (not simple at all, but there is a chance).
- In one of my projects, I used MBP at N terminus and His at C terminus, than you can combine the properties of both :-).
- If you do not want to design a new vector, you can also try to reduce the conc. of IPTG, I also used 16°C for some protein and it helped. But prolong the cultivation after induction to 18h min.
- anyway there is much more you can try, just write some more info about your protein. Size, AA sequence, origin, PI,..
BR
Hi there
you can solublize the protein by sonication and agitation, because the protein is in inclusion body in this state.
good lock!
Hi Joyleen Maryann Fernandes You may try chaperone based procedure to get more soluble protein DOI 10.1186/1472-6750-7-32
DEar Joyleen
can you provide us some more information about your protein and the domain that you cloned and expressed?
Is it a membrane or soluble protein?
Is it contain cysteines?
Did you cloned the full lenght protein or just a domain?
Best regards
Manuele
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