Question on recombination in E.coli and in baculovirus

[Kay Schink]

DK wrote:
> 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
> 

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} }