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

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

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Subject: Re: Protein expression in Minimal media (M9)
From: jwallen {- at -} CSB {- dot -} WFU {- dot -} EDU jwallen {- at -} CSB {- dot -} WFU {- dot -} EDU
Date: 2007-04-19

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Subject: Re: Protein expression in Minimal media (M9)
From: Cynthia Kinsland clk10 {- at -} CORNELL {- dot -} EDU
Date: 2007-04-19

Subject: Re: Protein expression in Minimal media (M9)
From: rgccri {- at -} CID {- dot -} CSIC {- dot -} ES rgccri {- at -} CID {- dot -} CSIC {- dot -} ES
Date: 2007-04-19

What we usually do is to grow cells in this media:

400ml H2O milli-Q Autoclaved
120ml AAmix (5X) Filtered
60ml M9 (10X) Autocl.
6ml TES (100X) Filtr.
12ml Glucose 20% Filtr.
0.6ml MgSO4 (1M) Autocl.
0.18ml CaCl2 (1M) Autocl.
1.2ml Biotine (5mg/ml) Filtr.
0.6ml Thiamine (10mg/ml) Filtr.
0.1 ml Met (50mg/ml).
x ml antibiotic
(2 x 4)ml pre-cultures in LB + antibiotic

Then grow until the right OD (or just a little bit less) is achieved, and
add to the culture:

1.5ml SeMet (50mg/ml) Filtr. (This will stop any inherent synthesis of
Methionine)
6ml Glucose 20%
60ml AAmix (optative)
0.6ml Biotine
0.3ml Thiamine

Then grow for half an hour more and then add IPTG.

With this we get 100% SeMet incorporation and good protein expression levels.

Good luck!
Raquel

> 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.
> *************************************************
>

Raquel Garcia-Castellanos, Ph.D.
Dept. of Structural Biology
Molecular Biology Intitute of Barcelona
(IBMB-CSIC)
08034 Barcelona (Spain)
Phone: +34-934006100 Ext. 269

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

Previous message:
Subject: Re: Protein expression in Minimal media (M9)
From: jwallen {- at -} CSB {- dot -} WFU {- dot -} EDU jwallen {- at -} CSB {- dot -} WFU {- dot -} EDU
Date: 2007-04-19

Next message:
Subject: Re: Protein expression in Minimal media (M9)
From: Cynthia Kinsland clk10 {- at -} CORNELL {- dot -} EDU
Date: 2007-04-19