Protocol Online logo
Top : Forum Archives: : Protein and Proteomics

unstable protein expression,pleeeeeese help - (Jun/08/2008 )

Hi all,
I am facing a difficult problem for last ten months.this is esat-6 protein of mycobacterium tuberculosis.So many papers have come out of its expression and purification. I selected this gene together with three novel genes for cloning,expression and purification.All the other 3 got expressed, this one hasnot. mad.gif

This gene is 288bp,which was cloned into pET32a with kpnI and HindIII sites.transformed into BL21DE3, BL21DE3 pLys S and JM109DE3 cells ,no expression!! IPTG conc-0.1mM,0.5mM,1mM and 2mM were tried.Temperatures 37,28 were tried.No result. unsure.gif

Some experts suggested pET28a, so again this gene cloned into pET28a with NdeI and HindIII.All the three strains above used.It got expressed, nice over expression blush.gif
Repeated no expression, all the standardisations,but no expression. glare.gif plasmid retransformed ,checked in 6ml LB with 25ul/ml kanamycin.-protein overexpressed.repeated from the same culture in 100 ml for purification-- no expression.
again tried in 6ml culture - no expression.

tried to reclone,even the PCR didnot worked at 72C (standardised temp,as earlier it was working at all temperatures between 62 and 72) ),worked at 62C (faint band ) ,cloning done, but no colonies.
after two weeks,same primer vials,and old reagents were used at 72C, PCR worked (sharp bands at correct position) ,cloning worked,colonies came, but plasmid size 150 bp less!!!
Glycerol stock used,protein expressed in 6ml LB. Next day, neither glycerol stock nor streaked plate culture(from glycerol stock ) didnt worked.
I know iam confusing you, but I cant more clearly express my situation than this,
Hope anyone could help.Some 2-3 vials of protein is with me,that too in PBS,can I elute it and use? but it was stored for 5 months.
Really i am tired, iam not doing this work in my institution, i have gone somewhere else for this part, I went for 3 months period, now its really 11 months.
But I cant leave all my trials like that,hope someone could help

-niveda-

QUOTE (niveda @ Jun 8 2008, 04:44 AM)
Really i am tired, iam not doing this work in my institution, i have gone somewhere else for this part, I went for 3 months period, now its really 11 months.
But I cant leave all my trials like that,hope someone could help

My Heineken did not allow reading your post in full, so apologies if you have already done this:

I would suggest that you also express a protein in parallel that is easy to express. That way you will know if the problem is your technique somewhere or the protein/system itself. As for reading material: http://search.vadlo.com/b/q?sn=158621799&a...ssion&rel=0

..

-cellcounter-

It sounds like you might have a toxicity problem. What happens to the OD as you do your expression? If it doesn't increase consistently, this would explain the low yields, and the failure to express the following day. Basically, you have a low level of unintended (or leaky) expression.
Bill Studier (of pET fame) has written several reviews considering this effect. One thing he recommends is to plate out onto plates containing a bit of glucose, to completely shut off induction. You could also consider autoinduction media.

-swanny-

QUOTE (swanny @ Jun 9 2008, 09:14 PM)
It sounds like you might have a toxicity problem. What happens to the OD as you do your expression? If it doesn't increase consistently, this would explain the low yields, and the failure to express the following day. Basically, you have a low level of unintended (or leaky) expression.
Bill Studier (of pET fame) has written several reviews considering this effect. One thing he recommends is to plate out onto plates containing a bit of glucose, to completely shut off induction. You could also consider autoinduction media.


Hi,
thank you for this suggetion, i too feel the cell density is less in those expressing cultures, i will supplimentLB media with glucose, hope it is 1% glucose, and will let you know the result as soon as possible.Still another doubt persists, this is the esat-6 protein of m tuberculosis,w hich has been worked by several groups, they have not reported anything about such a toxicity,
anyway tahnk you very much for the suggestion.

-niveda-

QUOTE (niveda @ Jun 10 2008, 05:28 PM)
QUOTE (swanny @ Jun 9 2008, 09:14 PM)
It sounds like you might have a toxicity problem. What happens to the OD as you do your expression? If it doesn't increase consistently, this would explain the low yields, and the failure to express the following day. Basically, you have a low level of unintended (or leaky) expression.
Bill Studier (of pET fame) has written several reviews considering this effect. One thing he recommends is to plate out onto plates containing a bit of glucose, to completely shut off induction. You could also consider autoinduction media.


Hi,
thank you for this suggetion, i too feel the cell density is less in those expressing cultures, i will supplimentLB media with glucose, hope it is 1% glucose, and will let you know the result as soon as possible.Still another doubt persists, this is the esat-6 protein of m tuberculosis,w hich has been worked by several groups, they have not reported anything about such a toxicity,
anyway tahnk you very much for the suggestion.

Sometimes a lack of information in a paper does not mean that something doesn't happen!!! The other researchers could very well have had toxicity issues and simply not mentioned them...
Check out this paper by Studier. [attachment=4816:studier_et_al_2005.pdf] It's a few years old, so if you want his latest thoughts and research you might need to contact him directly. I understand he is planning on preparing a fresh paper with the most recent data, because by his own admission, he has told so many people in a large number of places slightly different recipes as his understanding has grown, he can't remember exactly what he has told to who!
A quick read of the paper suggests somewhere between 0.5 and 1.0% glucose works well, but you need to buffer the system to prevent acidification.

-swanny-

QUOTE (swanny @ Jun 10 2008, 06:32 PM)
QUOTE (niveda @ Jun 10 2008, 05:28 PM)
QUOTE (swanny @ Jun 9 2008, 09:14 PM)
It sounds like you might have a toxicity problem. What happens to the OD as you do your expression? If it doesn't increase consistently, this would explain the low yields, and the failure to express the following day. Basically, you have a low level of unintended (or leaky) expression.
Bill Studier (of pET fame) has written several reviews considering this effect. One thing he recommends is to plate out onto plates containing a bit of glucose, to completely shut off induction. You could also consider autoinduction media.



Sometimes a lack of information in a paper does not mean that something doesn't happen!!! The other researchers could very well have had toxicity issues and simply not mentioned them...
Check out this paper by Studier. [attachment=4816:studier_et_al_2005.pdf] It's a few years old, so if you want his latest thoughts and research you might need to contact him directly. I understand he is planning on preparing a fresh paper with the most recent data, because by his own admission, he has told so many people in a large number of places slightly different recipes as his understanding has grown, he can't remember exactly what he has told to who!
A quick read of the paper suggests somewhere between 0.5 and 1.0% glucose works well, but you need to buffer the system to prevent acidification.


Hi,
thank you for your reply, i tried to download this paper, it is getting downloaded with errors, it shows only some scattered alphabets, is it the problem with my connection, or something else, hope you will understand and help again

-niveda-

Depending on your browser, you might get a right-click option to "download linked file", or some variation of that. I often use that if a straightforward "dowload in new page (or new tab)" produces errors.

-swanny-