i am trying to over-express a recombinant protein in E. coli heterologous system. My protein is on pET 28 a(+) plasmid (T7 polymerase, kanamycin resistant). I inserted this vector in E. coli BL21 (DE3).
When i tried to over-express my protein, i tested two optical density for the beginig of induction and three IPTG concentration distinct. Unfortunately, expression level of my protein was to low in induced samples and uninduced control shows higher leves than any other condition. My protein is about 43 kDa and seems hidrophobic (GRAVY index is -0.094)
The picture attached shows a western blot (anti-histidine tag as primary Ab):
-2: uninduced control O.D. 0,6
-3: O.D. 0,6 / 0,1 mM IPTG
-4: O.D. 0,6 / 0,5 mM IPTG
-5: O.D. 0,6 / 1,0 mM IPTG
-6: uninduced control O.D. 0,8
-7: O.D. 0,8 / 0,1 mM IPTG
-8: O.D. 0,8 / 0,5 mM IPTG
-9: O.D. 0,8 / 1,0 mM IPTG
As you can see in the picture, lane 6 correspond to an uninduced control.
Can someone give any advice how express my protein in the right way?
Thanks in advance.