On a less mundane tack, I have noticed that when one of my proteins
crystallized under very banal conditions (10mM NaHEPES 7.0, 1mM DDM, nothing
else), tiny gas bubbles were trapped in the needle shower. I suspect CO2 from
decarboxylation, but can anyone suggest other possibilities, or has anyone seen
this in their crystalizations? I have never heard of this, except in thawing,
x-ray damaged crystals.
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: Jacob Keller
To: CCP4BB@JISCMAIL.AC.UK
Sent: Monday, November 03, 2008 1:37 PM
Subject: Re: [ccp4bb] Semet in non-auxotrophic strains
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: CCP4BB@JISCMAIL.AC.UK
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
