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Protein Expression in SF9/hi5 cells


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#1 dasani

dasani

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Posted 22 February 2010 - 05:38 PM

Hi,
I'm trying to express a 30kD protein HIS tagged protein in SF9 insect cells. Previously, i have successfully expressed a very similar protein using invitrogen's bac-to-bac system, with great yield.

I have cloned my gene of interest into the pFASTBAC shuttle vector, and used DH10BAC to insert the gene into a bacmid (insertion verified via PCR). I have transfected in the bacmid, and produced both P1 and P2 viral stocks, several different times. The cells die (as expected) when I put the P1 or P2 virus on new cell stocks. The cells behave/look similar to my previous (successful) experiments.

HOWEVER, i can't seem to find the protein! I've assessed soluble, insoluble and media (captured/concentrated using TALON cobalt resin). I've tried two different antibodies with Western Blotting--- anti-HIS and anti-protein of interest - neither detect any signal (besides a few x-reactive bands). I have wondered if the protein was being rapidly degraded - does anyone know if this happens?? Based on the protein sequence, and previous experience with mammalian lines, i expect that the protein COULD be secreted into media....

To summarize: I'm not sure what steps to take next...
I have verified the sequence of the plasmid construct and verified the bacmid insert via PCR
I have tried to produce virus several different times, each with the same result
I have tried to find the protein in soluble, insoluble and media...
any suggestions would be greatly appreciated!!!!!!

#2 Holsten

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Posted 23 February 2010 - 09:28 AM

First, cells are not supposed to die after you apply virus stock, unless you give them too much virus!
Did you titrate your viral stocks?
Check the manuals for titration procedures and recommended MOIs.
Second- if your cells are dead- there shouldn't be any protein, do not spend your time looking for it!
If cells are alive you can see first protein in 24-36-48 h.p.i., depending on the size, small proteins are expressed quicker and in larger amounts.
Third, your protein will be in the medium ONLY if it has signal peptide for secretion (like antibodies) or in case if there is a significant overexpression!
Good luck!

Edited by Holsten, 23 February 2010 - 09:34 AM.





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