Re: [ccp4bb] S-S in EC

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

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Subject: Re: Protein interface prediction tool?
From: Andrew Orry andy {- at -} MOLSOFT {- dot -} COM
Date: 2007-11-29

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From: "Sabini, Elisabetta" sabini {- at -} UIC {- dot -} EDU
Date: 2007-11-29

Subject: Re: S-S in EC
From: Bernhard Rupp bernhardrupp {- at -} SBCGLOBAL {- dot -} NET
Date: 2007-11-29

Nonredundant summary - thx to all submitters:

1) disulfide was often present even after expression in a normal BL21 strain

2) try OrigamiB (DE3), Rosetta-Gami 2 from Novagen, are codon optimized and
have mutations in the thiorodoxin reductase and glutathione reductase
genes
- expressing from pET32 vectors which contain a thioredoxin fusion tag
- extra purification steps are generally needed to remove the thiorodoxin
after cleavage of the fusion tag
- level of expression achieved was not very impressive for E. coli,
but enough to work with for structural studies
- generally human, extracellular proteins
- as always, protein dependent
- Origami strains are somewhat slow growing and sick-seeming
do not grow to very high densities and have to be coddled somewhat
- They grow well enough in auto-induction media

- grow to OD around 1.0-1.1 at 37, cool flasks in ice bath to 20,
induce o/n with 30 uM IPTG at 18°C.
- some success with periplasmic export for disulfide bridge formation
using the OmpA leader sequence (not the PelB leader from the pET plasmids)

3) used the AD494 (trx) strain with good results

4) "Tuner" strain (also Novagen) and low levels of IPTG to induce expression

- led to expression of a protein with one disulphide bridge

5) try pichia.

br
-----Original Message-----
From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of
Bernhard Rupp
Sent: Wednesday, November 28, 2007 2:55 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] S-S in EC

Dear All,

I need to express a mammalian protein with S-S bridges and would hope
to talk off-board with a person who knows about thioredoxin strains ect that
might work.

Thx, br
-----------------------------------------------------------------
Bernhard Rupp
001 (925) 209-7429
+43 (676) 571-0536
br@qedlife.com
bernhardrupp@sbcglobal.net
http://www.ruppweb.org/
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CCP4bb navigation

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

Previous message:
Subject: Re: Protein interface prediction tool?
From: Andrew Orry andy {- at -} MOLSOFT {- dot -} COM
Date: 2007-11-29

Next message:
Subject: "Finding Chlorine" =)
From: "Sabini, Elisabetta" sabini {- at -} UIC {- dot -} EDU
Date: 2007-11-29