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. *************************************************
