Dear Crystallographers, I am overwhlemed with all of the responses--thank you all very much for your help. This listserve is a fantastic resource, and it is great that there is patience for all of the mundane methods questions! I will try to do my part to help out when I can.
Thanks, Jacob Keller ******************************************* Jacob Pearson Keller Northwestern University Medical Scientist Training Program Dallos Laboratory F. Searle 1-240 2240 Campus Drive Evanston IL 60208 lab: 847.491.2438 cel: 773.608.9185 email: [EMAIL PROTECTED] ******************************************* ----- Original Message ----- From: Cynthia Kinsland To: Jacob Keller Cc: [email protected] Sent: Monday, November 03, 2008 12:29 PM Subject: Re: [ccp4bb] Semet in non-auxotrophic strains You can express Se-Met protein in non-auxotrophs using metabolic inhibition (Doublie. Methods Enzymol. 276, 523-530, 1997 and Van Duyne et al. J. Mol. Biol. 229, 105-124, 1993). The Studier auto-induction paper (Studier, Prot. Exp. Purif. 41, 207-234, 2005) describes an auto-induction Se-Met medium that gives sufficient incorporation of Se-Met for phasing in non-auxotrophs. Good luck, Cynthia On Nov 3, 2008, at 12:56 PM, Jacob Keller wrote: Dear Crystallographers, I have been using an E coli strain called Tuner (DE3) pLacI, which is not auxotrophic for methionine, nor is there an auxotrophic analogue available. It may be that my protein can be expressed in other strains, but this one works well right now, and it is usually better to stay with what works well. Has anybody tried growing semet protein in non-auxotrophic strains, or are there tricks to using non-auxotrophs for semet protein? Thanks, Jacob ******************************************* Jacob Pearson Keller Northwestern University Medical Scientist Training Program Dallos Laboratory F. Searle 1-240 2240 Campus Drive Evanston IL 60208 lab: 847.491.2438 cel: 773.608.9185 email: [EMAIL PROTECTED] ******************************************* ____________________ Cynthia Kinsland, Ph.D. Cornell University Protein Facility Director 607-255-8844
