We've seen nice in vivo activity (on purpose) from proteins cloned under T7 promoters but transformed into non-DE3 cells. In fact, friends working with more zesty enzymes who wanted a more "tunable" in vivo assay have had to mutate the ribosome binding sites for proteins under T7 promotors to knock down expression in non-DE3 cells.
Sometimes it doesn't take much of a protein for its activity to have an effect! ===================================== Phoebe A. Rice Dept. of Biochemistry & Molecular Biology The University of Chicago phone 773 834 1723 http://bmb.bsd.uchicago.edu/Faculty_and_Research/01_Faculty/01_Faculty_Alphabetically.php?faculty_id=123 http://www.rsc.org/shop/books/2008/9780854042722.asp ---- Original message ---- >Date: Wed, 25 Jan 2012 09:48:55 -0800 >From: CCP4 bulletin board <CCP4BB@JISCMAIL.AC.UK> (on behalf of Chun Luo ><c...@accelagen.com>) >Subject: Re: [ccp4bb] Expression of Viral proteins for crystallography >To: CCP4BB@JISCMAIL.AC.UK > > Toxicity of target proteins in pET vectors can > manifest itself without DE3. Some people suggest E. > coli polymerases causes low level of expression. The > observed mutation rate is thousands of fold higher > than what the textbooks say. It's easy to tell > toxicity from other causes of heterogeneity. Less > than expected numbers of colonies from > transformation. The growth rates of cultures from > different colonies are quite different. Most > mutations are single nucleotide change (and single > amino acid change) that reduces certain > functionalities of target proteins. However the > mutations can be outside those functional domains. > In my experience with a few toxic proteins, the > mutations are evenly distributed, no over > representation of any mutation. Isolated wild type > clones generate mutants after transformation. > > Chun > > > > From: CCP4 bulletin board > [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of RubA(c)n > SA!nchez Eugenia > Sent: Wednesday, January 25, 2012 8:52 AM > To: CCP4BB@JISCMAIL.AC.UK > Subject: Re: [ccp4bb] Expression of Viral proteins > for crystallography > > > > Dear Gregory and Darren, > > Thank you for your answers. They have been very > useful. > > 2012/1/24 Gregory Verdon <gregory...@gmail.com> > > it possible that the protein is toxic (even when > slightly expressed from your possibly leaky pET > vector), so that e.coli select for mutations that > kill expression of your recombinant gene ... > > > > > > Gregory, the protein may be toxic, but I do not > think that could be the problem. BSJ cells are not > meant to be for expression, but for cloning, so they > are not DE3 lysogenic. That is, they do not have the > T7 RNApol necessary for the expression of the > proteins inserted into pET plasmids. > > In any case, thank you very much for you help. > > > 2012/1/24 Darren Hart <h...@embl.fr>: > > > I think the explanation is this: > > The source is natural viral RNA which is a > mixture of naturally mutated > > sequences (e.g. flu forms such a quasispecies) > > See: > > > http://www.virology.ws/2009/05/11/the-quasispecies-concept/ > > > > The pooled RNA has an average sequence that you > see when you sequence the > > pooled cDNA (individual mutations are hidden by > the averaging effect of > > having many sequences present). > > > > But when you clonally separate DNA molecules by > transformation (1 plasmid > > enters 1 cell to yield 1 colony), you see each > individual molecule > > represented 100% in the sequencing chromatogram > from the plasmid DNA that > > you have isolated from colonies. > > > > This is effect is commonly observed when > sequencing influenza virus isolates > > from patients. It will have nothing to do with > the E. coli strain. You can > > avoid it completely by using gene synthesis. > > > > Darren > > Darren, thanks to your information about the > quasi-species, now we are convinced that this may be > the problem. We have been thinking about this and we > have conclude that the average sequence may not even > exist and if it was, it might not be the active one. > That is to say, maybe we can not synthesize our gene > because we don't know whether the mutations are > needed or in any case what mutations would be > needed. Do you agree? And if I am right, is it the > only solution to try different mutants to get the > active one? > > Anyway, I think I would not need to synthesize the > gene because I can select one monoclonal colony and > delete the mutation by site-directed PCR > mutagenesis. > > Best regards,
