gst tagged protein expresion - (Feb/11/2007 )

Hi all,
I got a problem. and any of your suggestions will help.
I cloned 2 genes in pGEX, one of 10 KDa and another 130 KDa, so I expect proteins of sizes 36 and 156 KDa respectively considering the GST tag (26 KDa).
I sequenced both and seem to be ok, correct reading frame etc.
I tried the following conditions.
30C , 0.1 mM IPTG 0.5 mM IPTG 3 hr
37C , 0.1 mM IPTG 0.5 mM IPTG 3 hr
21C , 0.1 mM IPTG 1 mM IPTG Over night (15 hours)

In all cases IPTG added after growing cells at 37 until OD 0.6-0.9
and I am using Rosetta strain since this are human genes

Results: for the 156 protein.... nothing
for the 36 KDa protein. .. I can see a band that is induced but size is less than 30

I was mainly working with 20 to 50 ml culture to have enough extract (around 10 ml) to sonicate. smaller volumes froth too fast.

I would really appreciate any suggestions that you might have

cheers

I dont have any experience with rosetta, but...

have you run a total extract? Dont just run the soluble fraction.

sometimes its difficult to see expression because you have poor expression, and the protein as the same size as one of e coli. you can use a sds page gel with different % and try if you see the diference between before induction and o.n. total extracts.

I usually use an OD of 1.0. And check for tocicity issues, like poor growth. Maybe its better to use 2% glucose.

I have seen clones with expresion with a band with less then 30kDa, wich is the size of GST. There is a lot of possible explanations. But its best to try diferent colonies.

Thanks for replying Nori,
I ran both soluble fraction (after soication and centrifugation, and total extract before sonication, and in both cases I see this band below 30.
Grow seems to be ok, since I have a sharp increase of OD.
If I understand right.. the addition of glucose can improve induction? or its just for them to grow better?
Lookin carefully at the gels I have definitely induction of a protein around 26-30 KDA when I expect 37..
I am transforming again my construct just in case I have mistaken it with the empty vector

Thanks again

Glucose is used to prevent toxicity issues, because it prevents leakage expression before induction. But as you dont notice tocicity, you may not need it.

Its a good idea to double check your clones, and have another go.

You might want to consider to do a purification just to see if the column grabs something of your expected size.

good luck