Re: [ccp4bb] Weird expression behavior

- 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: Re: CNS to PDB format conversion
From: Eleanor Dodson ccp4 {- at -} YSBL {- dot -} YORK {- dot -} AC {- dot -} UK
Date: 2009-09-02

Next message:
Subject: Re: Weird expression behavior
From: Israel Sanchez israelsanfer {- at -} GMAIL {- dot -} COM
Date: 2009-09-02

Subject: Re: Weird expression behavior
From: Ezra Peisach epeisach {- at -} GMAIL {- dot -} COM
Date: 2009-09-02

Been there, done that, got the T-shirt.

I do not believe there is a protease like 3C in E. coli.

That said - do you have any rare codons in your protein - that might
cause the stall/termination in expression - leaving you a large amount
of tag. Have you followed through with purifictaion to see if you have
low levels of full length expression? If rare codons are a problem -
multiple vendors have cell lines [such as BL21(DE3) RILP, codonplus, etc.)

With regards to His tag causing inclusion bodies - there have been
publications regarding solubility issues w/ His tags (I think Helena
Berglund has a paper on this).

Have you considered (after taking rare codons into account), expression
with no tag at all? pET3, pET11, etc.

Ezra

Israel Sanchez wrote:
> Hello crystallographers in general and E.coli-protein producers in
> particular,
>
> I would like to share with all of you a strange behavior of two of my
> expression constructs, looking for some advice or just know if anybody
> has experienced something similar:
>
> The scenario is the following one, I am trying to produce a NTPase
> domain of around 20KDa of a human protein in E.coli. Initial cloning
> in a T7-based vector with a N-terminal-hexa histidine tags produced
> big quantities of an unfolded protein, in inclusion bodies. I tried
> all normal approaches to try to make the construct soluble: lowing
> expression temperature, lowing the concentration of IPTG, different
> growing mediums, different E.coli strains, ... no success.
> Then I decided to try some fusion-protein strategies, I cloned the
> same construct as a fusion protein with GST and MBP. Then, I could see
> a good soluble expression BUT only of the carrier protein (GST or
> MBP). Lowing expression temperature or lowering IPTG concentration
> does not produce any improvement. Both fusion-protein construct
> contain between the fusion partner and my protein a 3C site for
> cleavage with this protease.
>
> So, the question is, does E.coli may posses protease similar to 3C
> that may explain the self-cleavage? why my ribosomes are not reaching
> until the end of the construct?
>
> Thank you so much for your attention, any comment and/or suggestion
> would be highly apreciated¡¡¡¡
>
>
>
> --
> PhD. Israel Sanchez Fernandez
> EM-lab
> Departamento de Ciencia de Proteinas
> CIB-CSIC Madrid España
>

CCP4bb navigation

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

Previous message:
Subject: Re: CNS to PDB format conversion
From: Eleanor Dodson ccp4 {- at -} YSBL {- dot -} YORK {- dot -} AC {- dot -} UK
Date: 2009-09-02

Next message:
Subject: Re: Weird expression behavior
From: Israel Sanchez israelsanfer {- at -} GMAIL {- dot -} COM
Date: 2009-09-02