problem in expression of target protein

hi
i have cloned a 600 base pairs gene in pet blue vector
first screened the clone through blue white screening
i have confirmed recombinant by colony pcr
i have got amplification in colony pcr and positive result in restriction analysis also
i have transformed the recombinant plasmid in to expression host BL21
i am doing expression analysis by induction with IPTG
but the result is a small hair line band of the target protein which was similar in both induced and un induced cultures
please find me some solution for getting good potent expression
it is very impartant for me now
please help me
thanks in advance

QUOTE (janu @ Feb 5 2008, 12:25 AM)

hi
i have cloned a 600 base pairs gene in pet blue vector
first screened the clone through blue white screening
i have confirmed recombinant by colony pcr
i have got amplification in colony pcr and positive result in restriction analysis also
i have transformed the recombinant plasmid in to expression host BL21
i am doing expression analysis by induction with IPTG
but the result is a small hair line band of the target protein which was similar in both induced and un induced cultures
please find me some solution for getting good potent expression
it is very impartant for me now
please help me
thanks in advance

You might be having a toxicity problem. Check out William Studier's recent papers, including [attachment=4162:studier_et_al_2005.pdf].
In the meantime, re-transform your cells and plate out on media containing glucose (this will inhibit unintended induction caused by trace levels of lactose in the bacto-tryptone of your plates). The colonies you get should be quite large, compared to the usual size; if this is so, your problems have probably been because of leaky induction of the T7 RNA polymerase. Our lab has done a couple of test expressions using Studier's media and the effects are amazing compared to the standard system!

Basically, you want to totally suppress the induction of your target protein until your cell density is high. Studier's papers include recipes for rich media as well as fully defined media, including autoinduction media. These are good because you can inoculate the culture in the afternoon, and come back in the morning to a fully-induced culture. You will, however, need to to a time-course to determine the optimum length of the expt. If the protein is toxic, you might be better served to dod the expt during the day, with a short, sharp induction.

All the best!

Well, try to change your induction conditions. Each protein is a case...
Change IPTG concentration, incubation temperature and time (take several time points), etc.
There are several topics on the matter in this forum, try to look for some hints there.

QUOTE (swanny @ Feb 4 2008, 04:46 PM)

QUOTE (janu @ Feb 5 2008, 12:25 AM)

hi
i have cloned a 600 base pairs gene in pet blue vector
first screened the clone through blue white screening
i have confirmed recombinant by colony pcr
i have got amplification in colony pcr and positive result in restriction analysis also
i have transformed the recombinant plasmid in to expression host BL21
i am doing expression analysis by induction with IPTG
but the result is a small hair line band of the target protein which was similar in both induced and un induced cultures
please find me some solution for getting good potent expression
it is very impartant for me now
please help me
thanks in advance

You might be having a toxicity problem. Check out William Studier's recent papers, including [attachment=4162:studier_et_al_2005.pdf].
In the meantime, re-transform your cells and plate out on media containing glucose (this will inhibit unintended induction caused by trace levels of lactose in the bacto-tryptone of your plates). The colonies you get should be quite large, compared to the usual size; if this is so, your problems have probably been because of leaky induction of the T7 RNA polymerase. Our lab has done a couple of test expressions using Studier's media and the effects are amazing compared to the standard system!

Basically, you want to totally suppress the induction of your target protein until your cell density is high. Studier's papers include recipes for rich media as well as fully defined media, including autoinduction media. These are good because you can inoculate the culture in the afternoon, and come back in the morning to a fully-induced culture. You will, however, need to to a time-course to determine the optimum length of the expt. If the protein is toxic, you might be better served to dod the expt during the day, with a short, sharp induction.

All the best!

thank you
i will try this
but i am getting the expression is hair line band
is it possible to increse the expression
please help me
thank you

QUOTE (janu @ Feb 5 2008, 03:51 PM)

QUOTE (swanny @ Feb 4 2008, 04:46 PM)

QUOTE (janu @ Feb 5 2008, 12:25 AM)

hi
i have cloned a 600 base pairs gene in pet blue vector
first screened the clone through blue white screening
i have confirmed recombinant by colony pcr
i have got amplification in colony pcr and positive result in restriction analysis also
i have transformed the recombinant plasmid in to expression host BL21
i am doing expression analysis by induction with IPTG
but the result is a small hair line band of the target protein which was similar in both induced and un induced cultures
please find me some solution for getting good potent expression
it is very impartant for me now
please help me
thanks in advance

You might be having a toxicity problem. Check out William Studier's recent papers, including [attachment=4162:studier_et_al_2005.pdf].
In the meantime, re-transform your cells and plate out on media containing glucose (this will inhibit unintended induction caused by trace levels of lactose in the bacto-tryptone of your plates). The colonies you get should be quite large, compared to the usual size; if this is so, your problems have probably been because of leaky induction of the T7 RNA polymerase. Our lab has done a couple of test expressions using Studier's media and the effects are amazing compared to the standard system!

Basically, you want to totally suppress the induction of your target protein until your cell density is high. Studier's papers include recipes for rich media as well as fully defined media, including autoinduction media. These are good because you can inoculate the culture in the afternoon, and come back in the morning to a fully-induced culture. You will, however, need to to a time-course to determine the optimum length of the expt. If the protein is toxic, you might be better served to dod the expt during the day, with a short, sharp induction.

All the best!

thank you
i will try this
but i am getting the expression is hair line band
is it possible to increse the expression
please help me
thank you

OK, the issue I am concerned with is potential toxicity of the protein. Do the cells grow slowly before induction? Does anything bad happen to the cell density after you induce? If you do have a toxic protein, you need to make the expression as short as possible, but with as many cells as possible, so you get a decent total yield.

From your initial description of what you have done, it seems you haven't checked to see if your insert is in the proper reading frame in your expression vector. This might be something worth doing first.

QUOTE (wbla3335 @ Feb 5 2008, 05:25 AM)

From your initial description of what you have done, it seems you haven't checked to see if your insert is in the proper reading frame in your expression vector. This might be something worth doing first.

hi i have checked the reading frame by specific restriction analysis
thank you

problem in expression of target protein - (Feb/04/2008 )