is this protein too toxic to bacteria? - (Nov/04/2005 )

hey guyus~


i'm really struggling with the protein (His tagged)from Earthworm to express it in E.coli.

The vector system and E.coli strain im using are pET23a(+) and BL21DE3.

The problem is the vector containing inserts (i checked the sequence) has very little transformaty.
It was Really hard to get colonies.
(I tried to transformation with BL21DE3pLys .. but just the same.. )

Since i get just 2 colonies i checked the plasmid from the cell.

Then

I tried induction with 0.1/0.5/1 mM IPTG concentration , 25/28/30/37 temperature,
for 2hr/4hr/ overnight.

Nevertheless, i couldn't see Any distinct band.
I check it with Western blotting but the result was just the same.

I don't know what to do.
Is it because just my protein so toxic to E.coli?

(the protein is one of proteinase inhibitor) .

Any Idea? Thanks! :

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Welcome to the Club Bonnie I'm struggling too with my myccobacteria protein since one month

in theorie yes maybe your protein is toxic but since you already lowered the IPTG concentration and Temperature of growth you have been already quite smart

Basically got the same trouble than you with transformation of TunerpLac cells and TunerpLYS only pLAc cells worked probably my competent celles are not good enough !

Anyway I did as you did expression pilot experiments and failed so here is what I will try to do

1/ Shorten the protein size to be expressed, right now it's 20KDa I will change to 15KDa

2/ Change the region of the protein make one construct moved to NH2 terminal and one more upstream for another candidate.

3/ Screen more clones and use diffrent type of celles Origami, M15rep, Rosetta etc...

If everything fail then I will probably finfd another job

Keep going protein expression is a tough business ! I keep my fingers crossed for U

Pesji

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I had lots of problems expressing a bacterial problem (oh, I said problem, I meant protein - bit of a Freudian slip!!!) which was highly toxic. I found Origami B cells and the pET43 vector to be the best host/vector combination to obtain expression.


I was in the no-expression club for 8 months before success, so stick with it and good luck!

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bonnnie, this is probably a dumb question, but are you seeing a big band when you run just your lysate? are you sure it is the induction step that is the problem?

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It might also be a codon bias problem. The Rosetta strains (from Novagen) or the CodonPlus strains (from Stratagene) are designed to overcome such problems.

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hi..

my answer is 'no'
I already did 'western blotting' with anti-his primary antibody.
And.. i got no band on the right position .

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