Hi, I’ve received a number of concurring suggestions. Some have requested more detail about the experiments. Here are the details.
1. Cells: BL21(DE3) 2. Plasmid: pET28a (T7 promoter) 3. Media: TB 4. Protein: cytoplasmic 5. Expression: Grow at 37degC until OD600=0.6, cool to 20degC, induce with 1mM IPTG, grow <16hrs at 20degC, centrifuge. 6. Lysis method: Sonication via micro-tip/macro-tip. Small culture produces soluble protein where as large culture produces insoluble protein. We first thought it may be due to poor lysis, thus equivalent amount of cells from 3 and 500ml cultures were lysed with the micro-tip. Same results were observed. Best wishes, Reza Reza Khayat, PhD Assistant Professor City College of New York 160 Convent Ave, MR-1135 New York, NY 10031 (212) 650-6070 rkha...@ccny.cuny.edu<mailto:rkha...@ccny.cuny.edu> On Mar 19, 2015, at 11:15 PM, John Fisher <johncfishe...@gmail.com<mailto:johncfishe...@gmail.com>> wrote: Hi Reza. Clearly nobody needs to know anything about what protein you are specifically working on; that being said, in order to avoid a potentially endless email string of expert advices, please include everything detail-wise regarding your expression system, culture conditions, induction, and lysis method for BOTH the 3 ml and 500 ml expression trials. You will get, I imagine, amazing advices likely specific enough to solve the problem without your having to chase your tail. Best, John Fisher John C. Fisher, M.D./PhD On Thu, Mar 19, 2015 at 2:33 PM, Reza Khayat <rkha...@ccny.cuny.edu<mailto:rkha...@ccny.cuny.edu>> wrote: Hi, We can express quite a bit of soluble protein when growing 3ml cultures. However, the protein becomes insoluble (inclusion bodies) when we scale up to 500ml cultures. Has anyone experienced such a problem, and found a solution to it? Thanks. Best wishes, Reza Reza Khayat, PhD Assistant Professor Department of Chemistry City College of New York New York, NY 10031 http://www.khayatlab.org/ 212-650-6070<tel:212-650-6070>
