Hi, Cultures are being properly cooled prior to induction (water bath supplemented with ice).
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 From: lieh low [mailto:liehy...@gmail.com] Sent: Friday, March 20, 2015 8:06 AM To: Reza Khayat Cc: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Protein expression Reza, someone might have mentioned this, it takes longer time to cool down larger culture, we turn down the temp of the shaker when OD is about 0.4. For some protocol, we even use ice to cool the flask down before induction. You might also want to consider a lower induction temp, like 16degC. Maybe your protein is just temp sensitive. ray On Fri, Mar 20, 2015 at 6:56 AM, Reza Khayat <rkha...@ccny.cuny.edu<mailto:rkha...@ccny.cuny.edu>> wrote: 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<tel:%28212%29%20650-6070> 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>
