hi i am working on crystallisation of a 42 kDa his tag protein but,my protein gets precipitated at higher concenrtration till now i have not tried any salt to stabilise the protein,i am just confused what to do???should i go for higher salt concentration or anything alse.
-----Original Message----- From: CCP4 bulletin board on behalf of Raphael Gasper Sent: Tue 2/24/2009 9:56 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Off topic: Mammalian gene expression in E. coli This website is quite useful (via Expasy): http://gcua.schoedl.de/ Raphael Mo Wong schrieb: > Thanks for the reply. > > I've checked my sequence for rare codons; however, what would be > useful to a pseudo-molecular biologist like me is a web server which > will look at your input DNA sequence and guesstimate the success of > expression in E. coli (i.e., consider codon frequency). Does one > exist? Even better, a web server that offers the choice of different > commercial forms of E. coli (i.e., Rosetta and Codon+)? I tried > Googling this a few days ago and didn't find anything too useful? > > Cheers! > > On Tue, Feb 24, 2009 at 11:00 AM, <ar...@xtals.org > <mailto:ar...@xtals.org>> wrote: > > Hi, > > The question you have to ask yourself first is - does my gene actually > *have* the rare codons that you're trying to avoid? Experience > shows that > usually it's not single codons that are a problem but pairs or > triplets of > rare ones. If your gene does not have obviously bad codon > combinations you > still may derive a benefit from codon optimization and especially from > mRNA structure shuffling. There are articles out there that attempt to > present statistically significant evidence and I tend to agree > with them - > the practice of optimizing codon usage AND mRNA structure is a > good and > useful one. > > >From what you describe, you're working with a kinase. It is not > at all > uncommon to have toxicity of kinases for E. coli. Additional issues > include heterogenous phosphorylation if the kinase is normally > active or > can auto-activate in E.coli (classical example is PKA which can > phosphorylate itself in up to 30 places given the right conditions). > > Toxicity isn't difficult to spot - classical signs include > microcolonies, > heterogenous colonies, slow growth, plasmid instability and so on. > Even > with a native gene you would likely notice these symptoms to some > extent. > > Incidentally - toxicity usually equals folding, i.e. for a kinase > to be > toxic at least some of it has to be folded enough to work. This is > actually *good news* because toxicity can be combated on a > different level > than lack of folded expression. For example, co-expression with > phosphatases tends to work miracles for kinase-based toxicity. > > Finally, to answer your question directly - yes, i've seen several > cases > of proteins not expressing even with rare codon tRNA supplemented in > trans, but expressing well from optimized DNA. Again - optimized for > codons AND structure, as I've never separated the two processes. > > Synthetic DNA is cheap these days. If you can afford it - it's > useful to > try before taking the next step. In this case the obvious next step is > attempt at expression in insect cells - kinases usually work out > really > well in IC. > > Artem > > -- Raphael Gasper-Schönenbrücher Max-Planck-Institut für molekulare Physiologie Abt. für strukturelle Biologie Otto-Hahn-Straße 11 44227 Dortmund, Germany Tel: +49/231/133-2121 Fax: +49/231/133-2199
