toxic proteins in insect cells

Hi everyone,

We have a couple of cases of proteins that seem to be toxic when expressed in insect cells (using baculovirus) - we get very low transfection efficiencies and inefficient virus production despite the controls working fine.  In one case we got around this by incubating the cells for a much longer time period after transfection (~8 days), and then we did get some virus.  But I suspect one may select for low/not expressing virus using this method.

Does anyone else have any experience with this, or ways to deal with toxic proteins in insect cells?

 

thanks!

 

best,

peggy

 

Posted on 04-Jul-2012 17:18 CEST

Hi Peggy,

occasionally, we have constructs that do not lead to growth arrest after transfection of the bacmid even after extended incubation periods up to 10 days. The normal period is more like 5-7 days after transfection when cells stop dividing. Since we are splitting the cells daily and monitoring the duplication we can identify those difficult cases which generally do not show any expression.

The last case was a membrane-bound enzyme involved in lipid metabolism, where you can speculate that the lipid modification could affect the virus infection, maturation or release by the overexpressed enzyme but we don't have any data to show this. We tried several times with no success.

Maybe one could use known inhibitors of the protein or binding partners which could be co-expressed.

Hope that helps.

Best regards,

Hüseyin

Posted on 24-Jul-2012 10:35 CEST
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