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MBP fusion protein


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#1 singhp9

singhp9

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Posted 06 February 2015 - 03:44 PM

Hi,

 

I am trying to express a protein for structural studies in bacterial cells. The only tag that has helped solubilize the construct is His6-MBP dual tag at the N-terminus. When I do small scale expression test (2-3ml culture volume), I mostly see my fusion protein getting expressed (both in pellet and soluble fraction). However, when I scale up to 1-1.5L media per flask (4L capacity), I notice a significant amount of free MBP in my lysate and it also sticks to the resin (Ni-NTA and amylose). I induce the cells at same OD and grow them for same amount of time and I am using the same glycerol stock for expression.

 

Could someone please explain what is causing this difference in expression profile? Why am I getting so much free MBP? Is there a chance of early termination of protein translation. Is there a way for me to detect this possibility? And, yes I do use protease inhibitors in my buffers.

 

Thanks so much for your help and time!

 

Puja






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