If you try to lyse a cell pellet from 500 ml via sonication compared to the 3 ml that is a huge difference. In particular if you don't scale up the total volume. Say you lyse your 3 ml culture in 10 ml and your 500 ml in 20 ml. Your local protein concentration is perhaps 50x higher then, which might explain why your protein precipitates.
Try finding an alternative lysis method. Chemical or preferably cell disruptor. For my taste you are inducing to early. But here's a question. Did you run an induced versus uninduced sample to see what the final od600 is? I tend to induce at midpoint of the uninduced culture. Which easily could be od600>3 Jürgen ...................... Jürgen Bosch Johns Hopkins University Bloomberg School of Public Health Department of Biochemistry & Molecular Biology Johns Hopkins Malaria Research Institute 615 North Wolfe Street<x-apple-data-detectors://4>, W8708 Baltimore, MD 21205<x-apple-data-detectors://5/0> Office: +1-410-614-4742<tel:%2B1-410-614-4742> Lab: +1-410-614-4894<tel:%2B1-410-614-4894> Fax: +1-410-955-2926<tel:%2B1-410-955-2926> http://lupo.jhsph.edu<http://lupo.jhsph.edu/> On Mar 20, 2015, at 06:57, Reza Khayat <[email protected]<mailto:[email protected]>> 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 [email protected]<mailto:[email protected]> On Mar 19, 2015, at 11:15 PM, John Fisher <[email protected]<mailto:[email protected]>> 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 <[email protected]<mailto:[email protected]>> 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>
