[ccp4bb] Protein expression in Minimal media (M9)

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CCP4bb <-- 2007 <-- April 2007 <-- 18 April 2007

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Subject: Re: protein or salt?
From: Frank von Delft frank {- dot -} vondelft {- at -} SGC {- dot -} OX {- dot -} AC {- dot -} UK
Date: 2007-04-18

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Subject: Re: Protein expression in Minimal media (M9)
From: "Richard P {- dot -} Grant" rpg {- at -} USYD {- dot -} EDU {- dot -} AU
Date: 2007-04-18

Subject: Protein expression in Minimal media (M9)
From: mshah {- at -} HWI {- dot -} BUFFALO {- dot -} EDU mshah {- at -} HWI {- dot -} BUFFALO {- dot -} EDU
Date: 2007-04-18

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 <-- 18 April 2007

Previous message:
Subject: Re: protein or salt?
From: Frank von Delft frank {- dot -} vondelft {- at -} SGC {- dot -} OX {- dot -} AC {- dot -} UK
Date: 2007-04-18

Next message:
Subject: Re: Protein expression in Minimal media (M9)
From: "Richard P {- dot -} Grant" rpg {- at -} USYD {- dot -} EDU {- dot -} AU
Date: 2007-04-18