Re: [ccp4bb] Off topic: Mammalian gene expression in E. coli

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

Previous message:
Subject: Re: Off topic: Mammalian gene expression in E. coli
From: Mo Wong mowong22 {- at -} GMAIL {- dot -} COM
Date: 2009-02-24

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Subject: Re: Off topic: Mammalian gene expression in E. coli
From: Raphael Gasper raphael {- dot -} gasper {- at -} MPI-DORTMUND {- dot -} MPG {- dot -} DE
Date: 2009-02-24

Subject: Re: Off topic: Mammalian gene expression in E. coli
From: Partha Chakrabarti ppchak {- at -} GMAIL {- dot -} COM
Date: 2009-02-24

Just to add couple of things to what Artem said..

If it is something similar to a mammalian kinase or malaria protein for
example,

1. Recodonizaton can change expression from near zero to substantial
amounts, however,

a) ideally, it needs to be recodonized separately for each target expression
system (E. coli / yeast / IC)

b) Expression does not guaranty solubility.

One thing which sometimes works is coexpression of a phosphatase, if the non
phospho form is of any interest. Similar things have been done by Src
related kinases.. YopH or Lambda might be a good starting point. See:

http://www.ncbi.nlm.nih.gov/pubmed/16260764?ordinalpos=3&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_DefaultReportPanel.Pubmed_RVDocSum

HTH, Partha

On Tue, Feb 24, 2009 at 3:48 PM, Mo Wong wrote:

> I thought I'd post this to the CCP4bb, as judging by previous posts, it
> seems I could get some useful insight into my problem...
>
> This is question has probably been asked by people for a long as molecular
> biology has been around, but hopefully my question isn't a complete rehash
> of other peoples: I am trying to express a human protein in bacteria where
> the only modified amino acids are 3 phosphorylated serines. I’ve gone
> through the usual hoopla of trying to get it expressed in E. coli
> (Rosetta/Codon+ cells, varying IPTG, low temperature, etc). Sequencing
> confirms my insert is correct, but from coomassie gel inspection, I appear
> to get near zero induction (I need to do a Western to get a clearer
> assessment). I’ve heard about custom gene synthesis, and it appears Mr. Gene
> (https://www.mrgene.com/) would be a good avenue to look into as they
> optimize the ORF taking into account codon usage in E. coli (though I’m not
> sure they examine putative mRNA substructure formation like some companies
> do). It’s only 49c per base pair, so doesn’t seem too cost prohibitive. My
> only concern is that if this protein is toxic, I could be wasting money.
>
> So I was wondering, has anyone seen the expression for a particular
> protein change from zero in Rosetta/Codon+ cells using "native" sequeneces
> to being largely overexpressed in BL21(DE3) cells using codon optimized
> sequences? For folks who have had a similar problem to the one I've
> described, would you recommend that I first try using a codon optimized
> sequence in E. coli over testing protein expression in yeast/insect cells,
> or the other way round?
>
> Thanks!
>

--
MRC National Institute for Medical Research
Division of Molecular Structure
The Ridgeway, NW7 1AA, UK
Email: pchakra@nimr.mrc.ac.uk
Phone: + 44 208 816 2515

CCP4bb navigation

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

Previous message:
Subject: Re: Off topic: Mammalian gene expression in E. coli
From: Mo Wong mowong22 {- at -} GMAIL {- dot -} COM
Date: 2009-02-24

Next message:
Subject: Re: Off topic: Mammalian gene expression in E. coli
From: Raphael Gasper raphael {- dot -} gasper {- at -} MPI-DORTMUND {- dot -} MPG {- dot -} DE
Date: 2009-02-24