After inducing with IPTG for protein expression, my cell pellet after centrifuga - (Jan/16/2011 )
I have been growing and expressing a certain membrane protein in the lab and just recently, our lab relocated to a different city. However, I have been experiencing problems with expression of that protein. I typically innoculate LB with expression vector from a frozen glycerol stock, add antibiotics, grow overnight and transfer to a larger volume for growth and induction of protein expression. I have recently noticed that after my cell pellet after centrifugation is "goey", smaller than normal and the supernatant has debris. I don't know what to make of it, or if I should even proceed with an SDS-PAGE experiment to test for expression. Does anyone have an idea of why the cells are acting this way???
I am thinking of doing a miniprep from the glycerol stocks I have in the hopes of sequencing the DNA to search for rare codons that might be affecting protein expression.
Hola, I think that the problem is that your culture suffers lysis because the big amount membrane protein that it produces. Unless I think, you are expressing a mammalian protein in bacteria and this donīt recognize the signal peptide itīs possible that the produced protein coud be inserted in the membrane and do it explode. Anyway, try to induce lightly,less inducer, less time and less temperature, abut 28šC and follow the grow measuring OD at 600nm, could occur that the culture stand stationary or grow, but if the OD falls, you have to harvest quicky, and centrifuge at high speed to pellet the membrane fragments floating.Buena suerte