[ccp4bb] Protein expression in Minimal media (M9)

- Protein crystallography

Main steps:

- Protein purification
- Crystallisation

Special:

- Programs for crystallography
- X-ray detectors

Basic tutorials:

   - Chemistry
   - Protein
   - Peptide
   - Amino Acids

Xtal community:

- CCP4BB

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CCP4bb <-- 1999 <-- November 1999 <-- 30 November 1999

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

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

CCP4bb navigation

CCP4bb <-- 1999 <-- November 1999 <-- 30 November 1999

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