Hi, Well, sometimes proteins don't express even though they are from the same family and their "cousin" behaved very well. I found the followings helpful: 1. forget about the concept "this special strain *should* work because it is a Rosseta/ pLysS etc." Sometimes it's a long mRNA, sometimes a rare codon, sometimes it's an additional disulfide bond. If you are happy with the inclusion bodies protocol (and you know your protein is active later), just scan additional strains that you have in the lab (I like the BL21 DE3 BLR, which are specialized for long mRNA, but I found them useful in "regular" genes as well). 2. Scan "high" temperature (37C) in different time scales - maybe your cells need overnight expression instead of 3/4/5hrs? Or maybe the protein undergoes degradation overnight, so it is better to grow it for 3hrs only? 3. Change the medium to 2XYT or TB, and increase IPTG concentration if you work with a pET vector. 4. Try inducing in OD=1 instead of 0.6. 5. Add glucose (0.1%, but you can optimize) to the media to repress leakiness of a toxic protein. 6. Look for "autoinduced media" protocol. The additives there can make a difference between none to a few milligrams/liter. 7. Some mutations occurred in the expression vector. Re-clone to a fresh plasmid (same/ another pET vector/ vector with a different inducer, e.g. arabinose). 8. From my experience, sometimes E.coli get along with non-optimized eukaryotic genes even better than the optimized sequence. I would go to additional optimization as one of the last options. 9. A mammalian homolog? Or try expressing in lower temperature (17/20/23C) to get a soluble protein? (I know sometimes this is not an option).
A starting protocol would be several different strains (everything you and your neighbors have in the labs, basically), in 2XYT + 0.1% glucose, induction in 0.5-1mM IPTG, then grow 3hrs/5hrs/overnight in 37C. Alternatively, 2XYT+autoinduced media + 0.1-0.2mM IPTG when OD=0.6 to "boost" expression even further (and different times as well). Good luck :) Amit Dr. Amit Meir Institute of Structural and Molecular Biology Department of Biological Sciences Birkbeck College Malet Street London WC1E 7HX UK
