Re: [ccp4bb] Protein expression in Minimal media (M9)

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Subject: Re: Protein expression in Minimal media (M9)
From: "John A {- dot -} Newitt" newittja {- at -} COMPUSERVE {- dot -} COM
Date: 2007-04-19

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Subject: Re: Protein expression in Minimal media (M9)
From: Eric Toth etoth001 {- at -} UMARYLAND {- dot -} EDU
Date: 2007-04-19

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: etoth001@umaryland.edu
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:CCP4BB@JISCMAIL.AC.UK] On Behalf Of
mshah@HWI.BUFFALO.EDU
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.
*************************************************

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CCP4bb <-- 2007 <-- April 2007 <-- 19 April 2007

Previous message:
Subject: Re: Protein expression in Minimal media (M9)
From: "John A {- dot -} Newitt" newittja {- at -} COMPUSERVE {- dot -} COM
Date: 2007-04-19

Next message:
Subject: Post doctoral positions in Structural Immunology
From: Steve Almo almo {- at -} MEDUSA {- dot -} BIOC {- dot -} AECOM {- dot -} YU {- dot -} EDU
Date: 2007-04-19