Re: [ccp4bb] Expression of large proteins in E. coli

- Protein crystallography

Main steps:

- Protein purification
- Crystallisation

Special:

- Programs for crystallography
- X-ray detectors

Basic tutorials:

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   - Peptide
   - Amino Acids

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

Previous message:
Subject: Re: CCP4BB Digest - 12 Jan 2010 to 13 Jan 2010 (#2010-12)
From: Ho Leung Ng ho {- at -} CONFOMETRX {- dot -} COM
Date: 2010-01-13

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Subject: Re: Expression of large proteins in E. coli
From: Tuomo Glumoff tglumoff {- at -} SUN3 {- dot -} OULU {- dot -} FI
Date: 2010-01-14

Subject: Re: Expression of large proteins in E. coli
From: Will Stanley will {- at -} CYLLENE {- dot -} UWA {- dot -} EDU {- dot -} AU
Date: 2010-01-13

Hi Nick,

A couple of other suggestions:

(1) Try an E. coli K strain (e.g. JM109). They're different in
glucose metabolism (JN Phue et al, 2005, Biotechnol Bioeng 90:805–820)
and in my experience tend to grow more slowly - which seems to help
improve yields of large constructs.

(2) Optimal oxygenation also seems to be very important in producing
large constructs. Not sure what vessels you're using but Fernback
flasks work well for me. Or a fermenter might solve all you problems.

Good luck,
Will.

> Hello All,
>
> I apologize for the non-CCP4-related query. I have been working for
> several weeks now trying, with limited success, to express some very
> large proteins (ranging from ~100 to 180 kDa) from pET15b in E. coli.
> "Limited success" means I have expressed enough soluble protein to see
> on a gel, but not enough to purify. I have tried the obvious tweaks -
> changing strains (BL21, BL21-star, Rosetta, pLysS), screening
> induction temperature (16 to 37C), [IPTG] (0.3-1.0mM). I am in the
> process of subcloning into vectors for (1) SUMO fusion and (2)
> periplasmic expression (pET26b). I get the sense from digging through
> the literature that high level expression of large proteins depends
> mostly on the individual protein and I will ultimately have to look
> for homologs. But, this is my first experience expressing such large
> proteins and I am curious to know if anyone out there has some magical
> trick they wouldn't mind sharing.
>
> Thanks in advance,
> Nick
>
> -----------------------------------------
> Nicholas R. Silvaggi, Ph.D.
> University of Wisconsin-Milwaukee
> Department of Chemistry and Biochemistry
> 3210 North Cramer Street
> Milwaukee, WI 53211
>
> Phone: 414-229-2647
> Email: silvaggi@uwm.edu

CCP4bb navigation

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

Previous message:
Subject: Re: CCP4BB Digest - 12 Jan 2010 to 13 Jan 2010 (#2010-12)
From: Ho Leung Ng ho {- at -} CONFOMETRX {- dot -} COM
Date: 2010-01-13

Next message:
Subject: Re: Expression of large proteins in E. coli
From: Tuomo Glumoff tglumoff {- at -} SUN3 {- dot -} OULU {- dot -} FI
Date: 2010-01-14