Dear Ankit

Do tuo know why your protein is toxic? Is it an enzyme? It has to bind cofactors?

Unfortunatelly there is not a universal solution to yuor problem.

Each protein has specific properties.. for example . .. in my past experiences. I was able to produce stable Mia 40 only combining mbp fusion and origami strains.. for

..sod1.. i needed periplasmic expression and zinc addiction to the media..while i was not able to obtain the clostridium toxin a catalitic domain in any e.

Coli strain or conditions but it was able

To early produce it using brevibacillus expression system

However To answer to your specific question about the e coli strains: .scarabgenomics (http://www.scarabgenomics.com/c-17-mds-42-combination-package-chemically-competent-cell-kit.aspx) developed an interesting e.coli deleting large part dna and i think they are focus also in protease deleted strains but probably othis strains are quite expensives.

One other possibility is use pcold expression vector (takara) that work well at 17c and it is possibile that in this conditions the protease has lower activity.

One other possibile solution is purify your digested domain determine the n terminal sequence of your framment and try to remove the protease digestion site by site directed mutagenesys.

There are a lot of possibile strategies.. and no one is universal.. you have to choose it on the basis of your protein properties... good luck. Manuele

Thank you so much dear Manuele. Actually the protein is toxic because whenever it is expressed it goes to the membrane and damages it. As the growth was very slow I think that the fully functional protein was expressed to some extent but when purified the protein was truncated. I can try your recommendations and thank you for the link to strains.

How much is lower the size? Did you check that the protein band is not a bacterial protein expressed at high levels? Maybe using the same strain transformed with the empty vector.

Could you check your protein on a western blot?

In this way you cold see if you have two different size of the same protein.

I am dealing with similar problem.

You can connect two fusion partners to your protein enabling affinity purification. One at N-terminus and the second one on C-terminus. Like MBP, His-tag, GST, FLAG-tag, etc. Then you use appropriate resins in columns (amylose, Ni-NTA, etc.) in serial loading/elution steps. In theory only full form protein should be eluted in the end.

Of course this strategy can be complicated if your protein oligomerizes. Then you could perhaps disrupt the mixed oligomers (full forms and truncated forms in oligomer) by moderate concentration of guanidinium hydrochloride.

There is publication about it from the early nineties with His-tag&MBP combination. But I can not find it anymore.

Dear Peter, Thank you so much for the information. Actually I am trying the same thing currently. Hopefully it works.