pET IPTG expression - Getting protein expression (May/16/2007 )
Hi all. I have been trying to get a lux-HIS-tagged protein expressed from E. coli BL21(DE3). However, I am having two problems:
1. When I compare my induced (IPTG added) and uninduced controls I am getting the same level of luciferase activity in the sonicated cell lysates. I did not expect this, as I thought I should have no protein expressed in th non-IPTG added cells. Any thoughts why I am getting protein expression?
2. I am trying to purify the protein with His-tag using S-agarose kit made by novobiogen. It seems that are mulipte attempts I am losing my protein before the final extraction. I collect my flow-through elutes and find that from the time I first apply my protein to the agarose, then to the first wash, then second wash, then elution I have less and less protein. By the final elution I have lost EVERYTHING! I am trying to be as gentle as I can (centrifuging at 500 rpm only, and letting the protein bind in solution at 75 RPM in a flacon tube or flask. I have also added in PMSF to prevent protein degradation. Any suggestions on what might be going on or help?
i also had problems with IPTG induction, so you can try to do some test expression in about 10 ml medium, and test different IPTG concentrations: from 0.2 to 1.5mM. and see which one works best. Also the temperature control is really important! Do some test first and then see which one works best for you!
And if not....try to use another induction, arrabinose
always worked for me....but yes you have to change the vector and the cells....sorry for that
As for Ni-column kits: i have only nightmares about them! They never worked for me, so better try a regular Ni-NTA column. Let the protein bind to it always at 4deg and for a long time!!!