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Expression of a possible toxic gene?


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#1 uncutlateralus

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Posted 30 November 2009 - 07:38 AM

I've been trying to express a protein in E.coli that may or may not be toxic. The gene was cloned into a expression vector (pET28a) with the signal peptide region removed and i'm yet to get any protein expressed from any standard DE3 strains (BL21, B834, C41, Codon plus strains, Tuner, etc). I tried to modify the conditions (temp, time of induction etc) with no luck what so ever. Before anyone asks i've also double checked the construct with restriction enzymes and PCR so i'm happy my plasmid is intact.

The nature of the protein is slightly dubious, all I can say is its fairly closely related to phospholipase A2 in snake venom and therefore I suspect it may be toxic to E.coli, I monitored the growth curves of the induced E.coli culture and found that post induction (at around 0.6 O.D) the culture stagnated reaching only 0.7 compared to a non-induced protein growth curve which reached 1.4 in the same time frame.

However my supervisor has told me that all induced cultures stop growing post induction (little change at O.D 600), I guess normally its something I would never have bothered to check but i'm quite supprised that this occurs. Any ideas?


Any advice would be much appreciated, cheers!

#2 aimikins

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Posted 30 November 2009 - 08:58 AM

hmmm....sounds like it may be toxic.

the only thing I can suggest is to incubate at a much lower temp (if you haven't already) down to RT.

also, if it's being produced at all, you'll see it before the culture dies. have you tried a quick-and-dirty prep with SDS page of culture fractions? this will tell you if it's produced at all. I would try many timepoints, including a pre-induction sample for comparison. even if you don't see a band, if it's toxic in small amounts you may need to do a Western or something on your extracts.

PCR and RD notwithstanding, I'd take a few days and get it sequenced. you could have a point mutation or a frameshift or something that's hindering transcription

good luck
"it is a miracle that curiosity survives formal education" -A.E.

#3 smu

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Posted 30 November 2009 - 09:00 AM

Have you checked both soluble and insoluble fractions? And/or do you have an antibody that you could use to determine if it's being expressed?

#4 uncutlateralus

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Posted 30 November 2009 - 09:12 AM

Thanks for the advice guys,

aimikins, good idea with the quick dirty preps, I check my samples on SDS PAGE after about 3 hours induction using a cracking buffer. But i will have a check after half a hr next time at a much earlier time-frame. Sequencing is not a option saddly because of the cost but I did manage to 'borrow' some high performance, proof-reading polymerase so I think its probably unlikely to be a frameshift or point mutation present.

I his-taged it to aid in purification but also so I could do a western to check it out (i have the antibody) but our development fluid has run out so I'll have to wait a while till the order forms go through to check that out. I've tried expressing at temperatures as low as 20 degrees aswell.

The biggest worry I have is that the growth stagnates immediatly after induction but my supervisor is convinced that this isnt a issue at all so maybe i'm just being stupid

cheers!

#5 smu

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Posted 30 November 2009 - 09:52 AM

I second the sequencing of the clone. I guess I don't normally think of sequencing as being expensive, though I suppose if you have to mail it out it would be a bit more. Still you have to think about the cost of everything else you're doing with your clone in the meantime. Factor in failed experiments, used reagents, and your labor, and the cost is probably miniscule. I have had clones made with high fidelity polymerase contain errors so if you're really having a problem with it then i would try to convince your advisor that it would be worth the cost.

Just my .02.

#6 aimikins

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Posted 30 November 2009 - 02:14 PM

yes. I know that it's not free, but in terms of time, money, and reagent it will save you, you'll certainly come out ahead in the long run. you can tinker with a wrong plasmid for weeks or months, and that's a much bigger waste IMO
"it is a miracle that curiosity survives formal education" -A.E.




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