insoluble protein fraction - (Aug/14/2009 )
I am expressing one protein and Its going in pallete. My protein is expressing with out induction.......
but the protein s going in pallete. Its with out any tag......\
I try to make it soluble by detergent but still it does not bind with the column.......
I am expressing it at 30 degree can you suggest me some different tips to make protien soluble while it express.
I shall be very thankful to you for your reply.
You should consider cloning into a tighter plasmid, like pET LysS or pET LysE. These inhibit unintended induction, and stop expression until you induce.
Alternately, have you heard of autoinduction media? It has glucose or glycerol to prevent unintended induction caused by trace levels of lactose in the media. Some groups are suggesting its use because the process of induction, when it does happen is much gentler, that is slower, than simply dumping in some IPTG.
I would also consider dropping the temperature even lower to 18 degrees.