Question on recombination in E.coli and in baculovirus

I am considering co-expressing three proteins from the same
baculovirus. For that, a transfer plasmid from Novagen is
available which has two polh promoters and two p10 promoters.
Each of the same promoters is found on different strand
(i.e., one strand has one polh and one p10 and another strand
has one polh and one p10) "to minimize recombination" according
to the company. Promoters are ~ 70 and 90 bp.

More than that, I might need to express two of the proteins
as fusions with the same small protein (162 bp). There is no
problem placing those on different strand.

Because I don't really know much about homologous
recombination in either system, I worry if there are caveats
related to recombination and resulting instability of the
1) transfer vector in E.coli 2) resulting baculoviral DNA
in the insect cells.

Please give opinions on potential problems with the approach.
I always, of course, can go the route of co-infectin with three
separate viruses, painful as it is.

Thanks,

Dima

In <%xGyj.88$[Only registered users see links. ]>,
DK <[Only registered users see links. ]> wrote:


I wouldn't worry about insect cells - given the time length of expression,
after infection with baculovirus (or you concerned about recombination
when you make the virus?).

In E. coli, if I understand you correctly, the repeats will be inverted
(the polh and p10 promoters are on opposite strands, so they must be
inverted with respect to each other, correct?)

I don't think you'll have major issues with stability, but if you do,
propagate the plasmid in an E. coli strain like STBL2. We have had
very good luck propagating retroviral vectors, and retrovirus clones
in this strain, and both of these types of clones have two copies of
direct repeat, the LTR (U3RU5), and also four copies of inverted
repeats - at the U3 and U5 ends. The direct repeats LTRs range in
size from about 300 bp to 1 kb, and the inverted repeats are 15 - 30 bp
in length.

Anyway, these constructions are completely stable in STBL2. I think
we originally obtained this from Invitrogen.

AC
--
Email: echo 36434455860060025978157675027927670979097959886449 930P | dc

DK wrote:

There was an article in Nature Methods some time ago that might answer
some of your questions. The authors describe a baculovirus system
suitable for the expression of whole protein complexes from one virus.
This system is based on Invitrogens BacToBac-System but was modified so
multiple transfer vectors can be integrated into the the bacmid
harbouring the baculovirus genome. While there seems little probles
associated with recombination in E.coli, even within the large
baculovirus genome, the authors describe some problems with the
stability of viruses if they are repeatedly overamplified. If this does
not work out, you could always generate an individual baculovirus
expressing each protein alone and coinfect cells with the same MOI.

Best wishes

Kay



@article{
Author = {Fitzgerald, D. J. and Berger, P. and Schaffitzel, C. and
Yamada, K. and Richmond, T. J. and Berger, I.},
Title = {Protein complex expression by using multigene baculoviral
vectors},
Journal = {Nat Methods},
Volume = {3},
Number = {12},
Pages = {1021-32},
Note = {1548-7091 (Print)
Journal Article
Research Support, Non-U.S. Gov't},
Abstract = {Elucidation of the molecular basis of
protein-interaction networks, in particular in higher eukaryotes, is
hampered by insufficient quantities of endogenous multiprotein
complexes. Present recombinant expression methods often require
considerable investment in both labor and materials before multiprotein
expression, and after expression and biochemical analysis these methods
do not provide flexibility for expressing an altered multiprotein
complex. To meet these demands, we have recently introduced MultiBac, a
modular baculovirus-based system specifically designed for eukaryotic
multiprotein expression. Here we describe new transfer vectors and a
combination of DNA recombination-based methods, which further facilitate
the generation of multigene cassettes for protein coexpression (Fig. 1),
thus providing a flexible platform for generation of protein expression
vectors and their rapid regeneration for revised expression studies.
Genes encoding components of a multiprotein complex are inserted into a
suite of compatible transfer vectors by homologous recombination. These
progenitor constructs are then rapidly joined in the desired combination
by Cre-loxP-mediated in vitro plasmid fusion. Protocols for integration
of the resulting multigene expression cassettes into the MultiBac
baculoviral genome are provided that rely on Tn7 transposition and/or
Cre-loxP reaction carried out in vivo in Escherichia coli cells tailored
for this purpose. Detailed guidelines for multigene virus generation and
amplification, cell culture maintenance and protein production are
provided, together with data illustrating the simplicity and remarkable
robustness of the present method for multiprotein expression using a
composite MultiBac baculoviral vector.},
Year = {2006} }

In article <fqgsep$m2i$[Only registered users see links. ]-marburg.de>, Kay Schink <[Only registered users see links. ]> wrote:


Thanks, Aawara and Kay!

My concerns about E.coli are greatly alleviated now. I will have to
look at Multibac cloning details to see if they have significant
repeats.

With regard to the stability of viruses in Multibac: that's probably
function of Bac-to-bac itself. I am moving away from it precisely
because of that - there is something funky about it that makes it
not as appealing as it sounds on paper. Even with one protein, I
reproducibly observe that, without plaque purification of an individual
expressing clone, expression level always significantly drops with
*every* amplification step. Which is weird because it does not
correlate with virus titers at all. And this happens to a significantly
larger extent than with "normal" viruses.

For those that are interested, there is "FlashBAC" viral DNA
from Oxford Expression. They basically cloned ORF1629
truncated viral DNA into a E.coli BAC vector. So you only need
to co-transfect it with transfer vector. No need to plaque
purify and no need for transposition before transfection - the
best of both worlds.

DK