Protein expression with IPTG - (May/16/2007 )

Hi,
I use a pET vector system where the expression of my protein of interest is regulated by the lac operon.
I want to express my protein in transformated E.coli in 100 ml of LB broth, so I need to add IPTG to the broth.
Does anybody know if this is possible? Or do I have to use an other method?
I think that I need to much IPTG for high expression of my protein of interest...

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Grow your cells in a nice rich medium, from an overnight starter culture, until you have an OD of 0.6 or so. Add IPTG to a final concentration of 0.5 to 1 mM. Grow for 3-4 hr at 37 C, then harvest your cells.
If you have problems of low solubility, try growing at a low temperature for longer, such as 6-8 hr at 30 C, or overnight at 25C.

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The concentration wise, I guess use the standard one at 1 mM will be fine.

Swanny's protocols are about the same as mine.

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hi

is your ecoli strain BL21(DE3) ? its better for pET vector system
u keep ur Ecoli containing the vector till it grows to an OD 600nm for 0.7-0.8 -usualy 1.30 to 2 hrs and then add IPTG from stock solution and then keep again in the shaker for expression.
u can see the details in the manual for pET vector system.

then do as others have suggested ,
Try in small cultures first and find the optimal conditions for the expression

like IPTG concentration, How many hours after IPTG induction, The temperature

all these variations will give you an idea of which the condition that is best for the expression for your protein

try 37 degree , 1mM IPTG 1,3,5,7 hours first
then change the IPTG from 0.1mM to 2,3 mM and see

by this time u might get an idea after you veiw the sds page of your total protein from the lysate. both soluble and insoluble fraction
if nothing till now try differnt temp at a IPTG concentration you think showing most concentration. 20,25,30 etc

all the best

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Thank you for the help.

We want to express the GFP gene in BL21, so we dont do a SDS-page afterwarts.

I only want to express a little GFP which i can observe with a fluorescence microscope.

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then its easier , u can check flurosence instead of doing SDS PAGE and checking --if you can quanitify the flurosence it will give u an idea which is the best condition
i think there are jounarls that have done GFP expression in Ecoli check them and see what the conditions were
so u can lessen your time on optimization


all the best

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as others said, u can better ur expression, besides those, i think rossetta competent cells instead of bl21 could give u better results.

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