his tag protein not expressing - I am stuck !!! (Nov/29/2005 )

my protein is 15-27 kDa (different mutants).

I have confirmed the DNA sequencing and its in frame, but still it snot expressing in BL-21 DE3 cells.

My cloning vector has T7 promoter.

i am generating different truncated mutants of the same protein using the same vectore, and my problem is some of them are expressing others NOT AT ALLLL.

can anybody help me please

thanks

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is there a trend? for example, if you are cleaving off more of the C terminal with each truncation, and you notice that from a certain length --> longer, the protein is no longer expressed, then perhaps you can troubleshoot

maybe the more complete forms include some additional tertiary structure that affects solubility, or causes a complex to form with some ecoli protein, or results in some sort of recognition sequence that is cleaved/degraded by the host...

have you noticed anything in particular that can separate the ones that express from the ones that don't?

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You are very right, there is a trend. let me give u some background.

The gene has an N-terminal extension (NTE) , C-terminal extension (CTE) + 4 motifs divided into 2 domains which are connected by a connecting peptide (CP).

so i have sequentially deleted NTE, motif I, motif II, CP, motif III, motif IV and CTE and generated 7 mutants+++++wild type protein.

mutants which r expressing well ,, are truncated NTE, motif I, motif II and CP

mutants which are not expressing at all,, are truncated motif III, motif IV and CTE.

this is still understandable as u had mentioned, trunacting more from C-terminus is somehow inhibiting expression but my problem is the wild-type protein itself is not expressing

and i fail to understand this, as i have seen the expression of the same gene in another vector, but for this mutation project , i had cloned the gene in a different vector pET 100 TOPO.

let me know your views and suggestions

Thanks,
jhil

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why did you change vectors, if it worked in another?

what about changes in protein solubility between one form and the next? have you messed with the imidazole concentration, and other tricks that can affect solubility during purification?

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Originally we got this gene cloned in a vector through one of our collaborators.

After receiving it, i had expressed the protein for some experiment using conventional purification methods , whhich are time consuming.

Later, we planned to generate 8-9 mutants of this protein and so we had to look for a system which can help us in purification and thus we had to shift it to another vector which has His Tag.

I have not yet purified these mutant proteins so no question of imidazole. after confirming my DNA sequence, i did not see any expression after transforming it to BL-21 De3 cells and thats where i am stuck.

Any suggestion is welcome.
Thanks

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well, I would recommend doing all those trouble-shooting tips in your manual...it is time-consuming, but if you need to get purification sometimes it's necessary. You can do small-scale batch preps and look at different fractions; that will tell you if it's a solubility issue or an induction issue, at least.

do you see a potential band for your POI in the crude lysate? what about in your wash fractions? perhaps you are losing the protein somewhere. If you don't see something promising in your lysate, then perhaps you need to alter induction conditions.

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Hi Jhilmil!

Well, actually, I've had nearly the same problem with pET-System and BL21-cells. But when I changed induction temperature, I could detect my protein.
It made also a big difference between culture size. I optimized my expression conditions in 50 ml - and while expressing with these optimized conditions in 1 L, no protein was expressed...

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