problem with expression using baculoviruses

[Curt Ashendel]

On 5 Sep 1995 15:26:49 GMT, 
Shethah Morgan  <@fs1.ho.man.ac.uk > wrote:

>I have been unable to express HLA-DQ glycoproteins using the 
>baculovirus/insect cell system (detection by Western blotting). I 
>inserted the genes into pAcHLT-C and cotransfected with BaculoGold in Sf9 
>and High 5 cells with no luck.
>Does anyone have any explanations or advice? Look forward to some help.

A little more background would be helpful. Some thoughts...

Have you ever got a co-transfection to work? Perhaps there is 
something fundamentally wrong with your cells, methods,  or reagents. 

Do you get virus when using this plasmid? If not it is a 
co-transfection or reagent problem. 

Have you eliminated the 5' untranslated sequence or have you checked 
it for stop codons? Such have been known to suppress expression. 

Are you sure the transfer plasmid construct is correct? This 
problem turns up more often than you might expect. 

Did you western blot whole cell homogenates to see if the protein is 
made in an insoluble form? 

Did you check for expression at various times between 30 and 48 hours, 
in case it is rapidly degraded? 

Did you clone the recombinant virus or not? If not, it is possible a 
non-expressing recombinant dominates the virus pool. 

Does your antibody detect only the carbohydrate? Insect cell 
glycosylation is not always 100% the same as in mammalian cells. 


Curt Ashendel
Purdue University
West Lafayette, IN
ashendel at aclcb.purdue.edu