Request for Baculovirus Transfer Vectors

igp at mail.nerc-oxford.ac.uk igp at mail.nerc-oxford.ac.uk
Tue Jan 31 13:03:20 EST 1995


In article <3gbder$9b2 at mserv1.dl.ac.uk> r-mehta at nimr.mrc.ac.uk (Raj Mehta) writes:


>I am looking for anyone who has any double (or triple or more) promoter
>vectors for baculovirus expression - such as pAcUW31 (Clontech) or pAcUW51
>(Pharmigen) etc. I would be grateful if anyone can provide me with the
>above. Also, at the moment I am co-expressing two sub-units of a
>heterodimeric protein by co-infecting the cells with two different
>recombinant viruses. Has anyone got any info on the merits of this approach
>as opposed to the double promoter transfer vector?

Dear Raj,

I'm sorry that I cannot offer to send you the dual promoter baculovirus 
transfer vectors but I can provide some information on their use.  
In our lab, we have expressed up to 5 foreign genes using a single recombinant 
baculovirus.

It will be quicker and easier to clone your 2 genes into seperate transfer 
vectors to generate single expression recombinant baculoviruses.  With only 
two recombinant viruses you should get most cells infected with both types of 
viruses (with a moi of 5 to 10 pfu per cell each) and hence both genes 
coexpressed within one cell.  However, theoretical and practical studies here 
have shown that the ratios of the two recombinant proteins synthesised within 
each cell will vary significantly, depending on the kinetics of infection.  
The use of a single dual expression recombinant baculovirus would provide more 
consistant levels of each protein produced within cells.

Another point to consider is that the amount of foreign protein synthesised 
varies greatly depending on the gene being expressed.  The use of 2 single 
expression recombinant baculoviruses will, to some extent, allow you to vary 
the moi of each recombinant baculovirus to vary the average ratio of expressed 
products.

The final choice will depend on the specifics of your research.  Hope this has 
been of some help.  Please contact me by email if you require further 
information.

NOTE: the above information represents my own thoughts and opinions only, and 
not those of  NERC , Oxford University or the QDPI.

with regards

Ian Polkinghorne   (POLK at molbiol.ox.ac.uk)
NERC Institute of Virology and Environmental Microbiology
Mansfield Rd   Oxford  OX1 3SR
England

 



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