I cloned a gene in pET28a(+) vector and transformed in E. coli BL21 for expression. After IPTG induction and SDS PAGE analysis I am getting 3 bands that seem to be induced together instead of one with respect to control. The band of my interest also seems to appear but it is not a very thick band which is usually seen after IPTG induction. What can be the reason?
Gene size: 1280bp
Restriction sites: BamHI , XhoI
Protein Size: 48.32kDa
Induction Condition: 1mM final IPTG concentration, 8hrs, 25°C
Hello there,
It could be possible that the protein of interest is been cut by proteases in E. coli. That is why very little intact protein is been detected with the corresponding MW, while the other two bands may correspond to protein fragments. I would suggest to add protease inhibitors.
Hi Sagarika,
As Tatiana says you are probably looking at some protease activity.
Another possibility is if you have some rare codons or RNA structures in your gene that cause translation to stall at certain points, leading to the ribosome detaching early and so generating truncated protein and poor expression levels.
I'm assuming that you used BL21 (DE3) cells not BL21, as you would not get any induction from pET28 in BL21 as they don't have the the IPTG inducible lacUV5 promoter expressed T7 polymerase conferred by the DE3 lysogen.
- Richard
Thank You Tatiana and Richard. I will follow up on your suggestions.
@Richard: I am using BL21 (DE3) cells only.
I am also having same problem. I used same vector and BL21 star (DE3) . Easiest way to confirm is to cut your expected induced band from gel and send it for LC/MS analysis or perform western blotting. LC/MS helped one of my lab mate. we also cant see expected induced band very prominent other bands are more thicker even in empty vector lane in gel.
Adding protease inhibitor before disruption of bacteria using sonication will help to reduce the degradation of expressed protein. 1mM PMSF will be a good start point.
The pET28a vector contains the T7 inducible promoter, which is one of the strongest promoters available and is used for large scale heterologous protein expression in E. coli. For most proteins, large scale expression can be achieved at 37C for 1-2 hrs and the E.coli maintains a good level of fitness until about 3-4 hrs of induction. Beyond this time, over-expression could lead to stress on the bacteria (T7, being a strong promoter) and this could explain the production of other proteins alongwith the intended heterologous protein being produced. These proteins could be some stress-related host proteins being produced by the bacteria in response to stress.
Are you running control also? its better to induce empty vector also to check for leaky expression as well. Then you can conclude from where you are getting extra bands,
Hi
Try to confirm the immunogenicity of the bands whether they correspond to your protein of interest or not by performing a Western blot using specific antibodies.
In case you do not have those antibodies, at least try a western blot with anti-His antibodies as the recombinant proteins in pET 28 are labelled with His tags at N terminal.
Once you confirm the immunogenicity of the recombinant protein, then you could try time dependant expression (from 2h to 8h) of that particular band of interest (analyzed by Western blot). Also try expression at lower temperatures (25 C to 35 C).
All the best
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