Hello, Sherif Arafa

First you confirmed the expression of protein through WB. or visually, after different induction method. then go ahead.

You might have chosen a low expression vector or placed the protein gene after a basal-expression promoter, not an induced one. Maybe you can induce the expression of your protein with the T7 promoter system.

Another problem could be the toxic effects of the recombinant protein to the host or a problem related to the codon bias.

I am using already T7 promoter

i dont think it is toxic because similar protien was expressed using same competent cell

Hello Arafa,

Here are some possibilities that could explain the problem you are facing.

1. When you transformed your plasmid into BL21 cells after that did you run an SDS-PAGE? If so, did it show adequate expression? Sometimes you have to start with a higher culture to get the right amount of expression.

2. Before heading onto purification, did you do a sup-pellet localization study? It could be that your protein is getting expressed but all of it is going into inclusion bodies and hence after purification you do not get any yield and thus no bands in SDS.

3. Sometimes, if your Ni-affinity binding is not strong enough, you lose most of the proteins as flow through or during washing when passing the solution through affinity column.

4. The last possibility I can think of is that you said you did colony PCR. Since we rub the desired colony directly into the PCR mix, some unattached inserts also gets inside the mix thus giving false positive results. I suggest you do a restriction digestion of the plasmid to check whether insert has properly ligated in the vector and send your sample for sequencing if you have not done it already.

Hope this helps. All the best for your work.

Ditsa