What we usually do is to grow cells in this media: 400ml H2O milli-Q Autoclaved 120ml AAmix (5X) Filtered 60ml M9 (10X) Autocl. 6ml TES (100X) Filtr. 12ml Glucose 20% Filtr. 0.6ml MgSO4 (1M) Autocl. 0.18ml CaCl2 (1M) Autocl. 1.2ml Biotine (5mg/ml) Filtr. 0.6ml Thiamine (10mg/ml) Filtr. 0.1 ml Met (50mg/ml). x ml antibiotic (2 x 4)ml pre-cultures in LB + antibiotic
Then grow until the right OD (or just a little bit less) is achieved, and add to the culture: 1.5ml SeMet (50mg/ml) Filtr. (This will stop any inherent synthesis of Methionine) 6ml Glucose 20% 60ml AAmix (optative) 0.6ml Biotine 0.3ml Thiamine Then grow for half an hour more and then add IPTG. With this we get 100% SeMet incorporation and good protein expression levels. Good luck! Raquel > I had a similar situation, i.e. the cells wouldn't grow past an OD of 0.1 > on > minimal media, but in my case I did get protein expression. The way I got > around it was as follows. I grew the cells up in minimal media plus the > amino acids that suppress methionine biosynthesis, plus L-methionine. For > whatever reason, the cells behaved much better in "not quite minimal" > media. > Then, when they were near 0.6 OD, I spun them down and resuspended them in > media with Selenomethionine instead of L-Met. It worked for me (i.e. > ~100% > SeMet incorporation, structure solved, etc.), but I only needed to solve a > growth problem, not an expression problem. It might work for you, and you > could try a dry run without burning up precious SeMet. > > _____________________________________________ > > > > Eric A. Toth, Ph.D. > Assistant Professor > Department of Biochemistry and Molecular Biology > Marlene and Stewart Greenebaum Cancer Center > University of Maryland School of Medicine > 108 North Greene St. > Baltimore, MD 21201 > > Email: [EMAIL PROTECTED] > Phone: x-410-706-5345 > Fax: x-410-706-8297 > > http://www.umaryland.edu/bmb/faculty/toth.html > > http://crystal.umaryland.edu > > > -----Original Message----- > From: CCP4 bulletin board [mailto:[EMAIL PROTECTED] On Behalf Of > [EMAIL PROTECTED] > Sent: Wednesday, April 18, 2007 10:34 PM > To: CCP4BB@JISCMAIL.AC.UK > Subject: [ccp4bb] Protein expression in Minimal media (M9) > > Hello everybody, > > Sorry for an offtopic question. I am trying to express a protein in M9 > minimal media for Selenomet incorporation. When grown in LB this protein > expressed very well and got good crystals. Diffraction was upto 2 A. I am > having a hard time expressing the same protein in Minimal media. It took > nearly 24 hours for the OD600 to reach ~ 0.4 before inducing with IPTG in > minimal media and eventually got no protein expression. It looks like the > cells are not growing or taking very long to grow. The cell line I am > using is RossettaBlue (DE3) and the plasmid is pET19b based (Novagen). > > It expressed poorly in BL21 (DE3) when grown in LB and thus decided to use > RosettaBlue (DE3). It worked very well in LB, but having a hard time > while expresing the same in minimal media using Rosetta Blue. Has anybody > tried expression in minimal media using Rosetta Blue cell line? I am > planning to try overnight induction. > > Any suggestions would be greatly appreciated. > > Thanks, > > Manish > > > > > > > > > ************************************************* > Manish B. Shah, PhD. > Postdoctoral Fellow > Hauptman-Woodward Medical Research Institute > 700 Ellicott Street > Buffalo, NY 14203. > ************************************************* > Raquel Garcia-Castellanos, Ph.D. Dept. of Structural Biology Molecular Biology Intitute of Barcelona (IBMB-CSIC) 08034 Barcelona (Spain) Phone: +34-934006100 Ext. 269
