Re: [ccp4bb] "inclusion body"

- Protein crystallography

Main steps:

- Protein purification
- Crystallisation

Special:

- Programs for crystallography
- X-ray detectors

Basic tutorials:

   - Chemistry
   - Protein
   - Peptide
   - Amino Acids

Xtal community:

- CCP4BB

CCP4bb navigation

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

Previous message:
Subject: Co-expression plasmids
From: "Mark J {- dot -} van Raaij" VANRAAIJ {- at -} USC {- dot -} ES
Date: 2008-04-02

Next message:
Subject: Re: Co-expression plasmids
From: Raji Edayathumangalam redayath {- at -} MAIL {- dot -} ROCKEFELLER {- dot -} EDU
Date: 2008-04-02

Subject: Re: "inclusion body"
From: Louise Major llm6 {- at -} ST-ANDREWS {- dot -} AC {- dot -} UK
Date: 2008-04-02

Dear Skivesh,
My favourite media is TPB - (paper is: Moore et al Protein expression
and purification (1993) 4: 160-163). I've had luck with this and
BL21 (DE3) Gold cells. I'm also a fan of cold shocking cells - after
initial growth at 37 deg C, put flasks in an ice water bath for 10
min, then add IPTG and tranfer to 16-25 deg C for expression. This
combination rescued a protein that behaved like ball bearings in all
other conditions. It always seems worth trying an array of strains
vs an array of media vs cold shock/heat shock/chaperones, and a bit
of tweaking of [IPTG]

Good luck,
Lou

At 11:06 02/04/2008, you wrote:
>Dear all,
>Sorry for the off-topic question...
>What can be done to avoid a protein going inside inclusion body.The
>gene is cloned in pET30a with C-ter his tag and expressed in
>BL21-DE3 from 37 to 18C for 3-4 hr with .5mM of IPTG,it is going to
>inclusion body.All suggestions are welcome.
>Thanx in advance.
>Shivesh

********************************************************
Louise Major
Centre for Biomolecular Sciences
North Haugh
The University
St Andrews
Fife KY16 9ST
Scotland

ph +44 1334 467247
fax +44 1334 462595

The University of St Andrews is a charity registered in Scotland : No SC013532

CCP4bb navigation

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

Previous message:
Subject: Co-expression plasmids
From: "Mark J {- dot -} van Raaij" VANRAAIJ {- at -} USC {- dot -} ES
Date: 2008-04-02

Next message:
Subject: Re: Co-expression plasmids
From: Raji Edayathumangalam redayath {- at -} MAIL {- dot -} ROCKEFELLER {- dot -} EDU
Date: 2008-04-02