Protien expression - (Jul/01/2005 )
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.
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.
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)?