improve pritein expression - (Apr/22/2005 )

my recombinant protein is about 100kDa. i induced it by using 0.5mM IPTG for 4 hours 37 degree. but the protein expression level is quite low. i know this protein is quite big. so how could i improve the expression? induce longer ? or anything else i can do?

Thanks a lot
cathy

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Further information is required to pin down your problem. I presume that you have added IPTG while inoculating the culture. IPTG should be added only after the culture has reached mid-log phase. Immediated induction results in diversion of cellular resources to expression of the cloned protein and thereby starves the process of cell divison. The way out is to allow accumulation of sufficient biomass and therafter induce the expression.

The highest IPTG concentration used is generally 1mM. So the amount used, 0.5mM, seems to be OK.

Another possible interferecne might be the growth temepature. When other parameters are optimal, induction at 37C results high rate of protein production. If the processing of your nascent protein into folded state is a tedious process then the folding will lag behind expression. The unfolded proteins aggregated and form inclusion bodies. In such cases, expression at 30C or lower temeparature lowers the expression rate and allows greater yield.

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thank you sarah.
i do add IPtg at log phase. lower temperature can make the protein more soluble and fold properly. what i concerned is that my protein is so big, after induction for 4hours the expression level is not that high compared the smaller protein, should i give it longer induction time to increase the expression?

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Hi,
Sharath recommendations are excellent. However, I have found expression at 20C to get better soulbility compared to 30C and I have expressed my cells overnight with no problems, so yes you can go ahead and induce for longer periods. Good Luck!

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hi
I am expressing a his6 tag vector (soluble protein) in bl21 and inducing the culture with 1mM IPTG and incubating at 37 for 3 hours. My problem is like i got the required protein band after purification with Ni agarose with a 20mM imidazole elution. but with it i can see a higher mol weight band. this is not a required band. I would like to know how can i be specific for the extration of my protien molwt is 22kd. I tried to do the expression at 37 or 4 hours and also 30 for 2 hours, but in vain.i also tried higher conc of imidazole(20,50,100,200mM) but 20mM i found is optimal, yes in this eluate,i could fine only my band of interest which is very faint.
I would like your suggestions.
thankyou

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one more question, i grow my cells at 37 before induction and then to the required temp..37 or 30. so do i need to grow the cells from the beginning in the same temp at which i am going to keep after induction.
thankyou

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Just an example so you could figure out to try.

I expressed a his-tagged protein normally by adding 0.1mM IPTG at mid logphase. Induction temp 30oC. Result was fine.

The same protein, after to incorportating a change (a mutant), did not express at the above condition (I just checked the soluble portion).

In the later case, the mutant, I could express it at lower temperature (27-28oC) but the induction should be in the early of the log phase (OD600 ~0.4) and NOT after the mid of logphase.

Sometimes understanding the nature of the protein you want to express may help to solve the problem.

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