Hi James, Just a thought or two after reading your notes. I don't exactly know what KRX cells are but I suspect (based on your mention of rhamnose) that they are cells in which T7 RNAP levels are under the control of a rhamnose promoter. If that is so, it would be important to add rhamnose during cell growth to prevent an leaky expression of your protein, which may be toxic to E. coli, as opposed to adding it during the stage of induction along with IPTG like you describe. Just to rephrase, add inoculum, antibiotics and rhamnose to TB, grow to the OD you desire, then add IPTG to induce protein expression. Often, toxicity effects are felt in larger cultures and not seen in 15mL or 40mL cultures.
Also, I have found that there are certain cases when it is better to use Luria Broth as opposed to TB. But that is not my primary concern in your case. My two cents! Raji On Tue, Jan 15, 2013 at 9:48 AM, Murray, James W <[email protected]>wrote: > Dear All. > > A question on protein expression. > We have been doing small scale test expressions in 15ml of terrific > broth+kanamycin using E.coli KRX cells in falcon tubes. On reaching > OD600 of ~0.6 we induced with rhamnose+IPTG and expressed for 4 hours > at 37 C. There is a big band corresponding to our protein in the > soluble fraction and not much in the insoluble fraction. However, on > scaling up to 1 L TB cultures (with same concentrations of kanamycin > and inducing with same concs of rhamnose+IPTG) we don't get strong > over expression. Has anyone else experienced problems when scaling up > expression? (and more importantly, solved them?) > > best wishes > > James > > > -- > Dr. James W. Murray > David Phillips Research Fellow > Division of Molecular Biosciences > Imperial College, LONDON > Tel: +44 (0)20 759 48895 -- Raji Edayathumangalam Instructor in Neurology, Harvard Medical School Research Associate, Brigham and Women's Hospital Visiting Research Scholar, Brandeis University
