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E.Coli ceased to express my target protein - (Oct/31/2008 )

Dear All,

I am trying to overexpress a human recombinant protein in E.coli. After screening for a suitable cell line for expression (RIL, Rosetta, Origami, Rosetta 2) I found that Rosetta 2 was able to overexpress neatly my target protein into inclusion bodies at various conditions, such as 37C/4h/1mM IPTG; ON expression @ 18C with 50uM IPTG and also autoinduction. A glycerol stock was prepared of these cells.
After using up all protein for refolding experiments I wanted to produce some more protein using the cells from the glycerol stock, however I only get a very (very) low yield of protein. So I transformed the vector again into competent cells, but there still is no expression. The original plasmids (pPROex) were sequenced. I performed cell PCRs, which showed that the vector is there in all cases. The LB was always from the same batch as well...
So basicly my conclusion is that the cells, for what ever reason ceased to overexpress THAT protein. I can use the cells for overexpressing other proteins...
I wonder if something like that has ever happened to anybody. Any suggestions are welcome.
Thanx!

-lost higo-

If the plasmid has not mutated, and the cells have not mutated, then there's something changed in your growth conditions, overexpression conditions, or purification conditions.

Is your frozen stock of transformed E. coli from a single colony isolate of the clone that at one time worked?

-HomeBrew-

QUOTE (HomeBrew @ Oct 31 2008, 12:08 PM)
If the plasmid has not mutated, and the cells have not mutated, then there's something changed in your growth conditions, overexpression conditions, or purification conditions.

Is your frozen stock of transformed E. coli from a single colony isolate of the clone that at one time worked?


I freshly transformed the plasmid, which i originally used and picked 15 colonies for test expression. None expressed, all of them carry the insert. I'll try to clone again into a different vector, since my only explanation is that the plasmid has somehow mutated..

-lost higo-

No -- I'm asking about the first transformation -- the one from which you got your clone that worked. Was the clone that worked a single colony isolate, and did you freeze it as one?

What I'm trying to discover is if your E. coli strain that expressed correctly was a spotaneous mutant that you happened to pick from a lot of possible (and presumably wild-type) candidates from the first transformation. What is the lineage of this strain -- and was a single colony isolate of it what your froze for your glycerol stock?

Clearly something has changed. Maybe you should re-sequence the plasmid contained within one of the strains that doesn't seem to work any more to insure that the plasmid hasn't mutated.

But, as I said before, if the E. coli hasn't changed, and the plasmid hasn't changed, and your original working line wasn't a spontaneous mutatnt that allowed high expression, then the only explanation left is that something external has changed -- a new batch of IPTG unintentionally made at 10x (or 0.1x), some buffer in your purification procedure has an altered pH or been re-made and is now different than before, stuff like that...

-HomeBrew-

I am still clueless. Waiting for the results from re-sequencing. Meanwhile I tried 0.1 , 1 and 10 mM of IPTG, different pH and recloning into another vector (pET28 derivative). Since I had 2 different clones which encoded either the full length or a 150aa truncated version, which both ceased to work I believe that something changed in the growing conditions, but I really have no clue what. I reached the point where I believe that changing to another expression system is the best solution.

-lost higo-

Hi

I know what ur talking about. After reading what u wrote i thought that it was me that writhing it.

It all happened to me too and no matter what i tried to do (like u) didn't help. i also did new transformation with the plasmid to

new cell froze it in glycerol- first time positive signal, but on the second time the expression was lost.

Sorry man but i don't know what had happened and no one else know of such a problem.

So what to do ? starting all over: new PCR product, new cloning, plasmid and cells.

by the way, i am working with pBAD vector and top10 or LMG cells (invitrogen)

good luck.


-amtash-