Protien expression - (Jul/01/2005 )

Hi,
I have cloned which is supposed to be a lipoprotien but it lacks the signal sequence. So, I cloned the whole gene into pQE30 vector and transformed in E.coli. I checked for the orientation by pcr and it is fine.

But when I induced with 1mM IPTG for 5 hours, I did not get any induction at the size of 43 KDa, instead some other protien at ~63KDa got induced.
I also looked in supernant, but I could not find any protien in supernant.

I don't know if my protien is expressed or is it present in periplasmic space.
Could someone help me?

Thanks i advance.
Shilpa.

Hello Shipa,

Have you checked your insert sequence by sequencing? well,if you have AntiHis monoclonal Antibody, you could check the western blot.if the protein is expressed in very low concentrations also, u will be able to detect the expression. This antibody is costly, but really helpful and can save lots of time .All the best.

Saikat Chakraborty
Mie University
Japan

What do you see if you run a gel of whole cell lysate (just take samples of your clone, induced and uninduced, put them directly into loading buffer, boil, and load)?