Expression of Viral proteins for crystallography

From: Rubén Sánchez Eugenia
Date: 24 January 2012 16:16

Dear everyone,

I am trying to clone a viral protein in the E. Coli BSJ strain and i am having some problems.

I start from the viral RNA carrying out a reverse transcription and PCR (RT-PCR) to obtain the protein cDNA. When I sequence this cDNA to check for mutations, there are no mutations. So the RT-PCR works fine.

Then, I digest the cDNA and I ligate it with a pET plasmid to transform the E. Coli BSJ strain. I get recombinant colonies (checked by colony-PCR) but when I sequence them I get various mutations (aprox. 2 miss-sense) on the inserted cDNA. Furthermore, these mutations are different among different transformations and even among colonies of the same plate (in the same transformation).

Maybe these mutations are produced by the cell (because of the lack of mutations in the cDNA) but these E. Coli clonning strains are supposed to be "optimized" to prevent the insertion of mutations. So I have no idea about what may be the problem.

I hope you could help me. Thank you.

Best regards,

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Rubén Sánchez

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From: Darren Hart


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

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Dr. Darren Hart,

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From: Jacob Keller


Inspired by the recent post about "quasispecies:"

I have been bothered recently by the following problem: why do species
of genetic uniformity exist at all (or do they?)? This first came up
when I saw a Nature paper describing live bacteria extracted from a
supposedly 250-million-year-old salt crystal whose 16S RNA was 99%
identical to marismortui bacteria (ref below). What? Are the bacteria
the same now as 250 million years ago? But there is a further
question: given the assumptions of evolution, why should there be any
bacterium whose genome is the same as any other, assuming that
equivalent codons are really equivalent (or at least roughly so), and
that even at the protein level, there is such a thing as "neutral
drift?" After all, we even see in our lab cultures that they (at least
e coli) mutate fairly frequently, so why is there such a thing as "e
coli" at all, at least at the nucleotide level? I don't think we
usually say that each bacterial species is totally optimized in all
its features, do we? Even assuming that every single protein must be
just so, shouldn't there be as many species of e coli as there are
possible genomes encoding the same protein set, i.e. some extremely
large number? Why is there any uniformity at all? Or IS there--maybe
the bacteria too are only quasispecies...? And maybe also...

JPK




Nature 407, 897-900 (19 October 2000) | doi:10.1038/35038060; Received
15 November 1999; Accepted 4 July 2000

Isolation of a 250 million-year-old halotolerant bacterium from a
primary salt crystal

Russell H. Vreeland1, William D. Rosenzweig1 & Dennis W. Powers2

Department of Biology, West Chester University, West Chester,
Pennsylvania 19383 , USA
Consulting Geologist, Box 87, Anthony, Texas 79821, USA
Correspondence to: Russell H. Vreeland1 Correspondence and requests
for materials should be addressed to R.H.V. (e-mail: Email:
rvreeland@wcupa.edu).

Top of page
Bacteria have been found associated with a variety of ancient
samples1, however few studies are generally accepted due to questions
about sample quality and contamination. When Cano and Borucki2
isolated a strain of Bacillus sphaericus from an extinct bee trapped
in 25–30 million-year-old amber, careful sample selection and
stringent sterilization techniques were the keys to acceptance. Here
we report the isolation and growth of a previously unrecognized
spore-forming bacterium (Bacillus species, designated 2-9-3) from a
brine inclusion within a 250 million-year-old salt crystal from the
Permian Salado Formation. Complete gene sequences of the 16S ribosomal
DNA show that the organism is part of the lineage of Bacillus
marismortui and Virgibacillus pantothenticus. Delicate crystal
structures and sedimentary features indicate the salt has not
recrystallized since formation. Samples were rejected if brine
inclusions showed physical signs of possible contamination. Surfaces
of salt crystal samples were sterilized with strong alkali and acid
before extracting brines from inclusions. Sterilization procedures
reduce the probability of contamination to less than 1 in 10 9.

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From: Stefan Gajewski


Rubén,

the previous answer probably addresses your problem accurately.

However. If your protein of interest modifies DNA/RNA, it is quite common that your pET constructs will mutate rapidly in E.coli. Lac operons tend to leak quite a bit, which is not enough to detect the protein prior to induction but can definitely drive the selection for mutated plasmids in E.coli. We saw that behavior in a nuclease cloned from homogeneous genomic DNA. Every colony had a different mutant when grown on LB.  Also mutants that do not appear to affect the enzyme activity judged by the structure. We assumed, that any mutation that slightly reduces the toxicity for E.coli will be selected for. So, if you experience the same issue again with synthetic DNA, or have the patience to repeat, try adding ~1% glucose to all media, liquid and solid. This will tighten up the Lac operon and worked well for our nuclease project.  You still have to sequence every batch you purify. pLysS cells can also work, but we still ended up adding glucose because it is cheap and doesn't cause trouble downstream.

Stefan

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From: Rubén Sánchez Eugenia


Dear Gregory and Darren,

Thank you for your answers. They have been very useful.

2012/1/24 Gregory Verdon 

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.
 

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,

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From: Chun Luo

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

 

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From: Phoebe Rice


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!

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Phoebe A. Rice