We have been expressing a soluble protein in sf9 cells after infection with recombinant baculovirus (constructed using the bac2bac system). We have been able to see expression of the protein based on western blot and silver stain (NiNTA purification) but the effect has been non reproducible. From 5 or 6 expression attempts, 2 gave the correct protein, the rest gave nothing. The baculovirus stock (both the P0, P1 and P2) appear to be active and lead to swollen cells and cell lysis after a few days.
Any ideas on what's going on? Is it worth going back and re-making baculovirus and a P0 stock? We have confirmed that the bacmid contains the gene of interest by PCR.
If you are getting the cell swelling, lysis, expression and sequence in bacmid, do not think that construct is a problem. Just check that cells and the backmid you are using are of good quality and no of cells and amount of backmid is proportional. In cells it happens some time. If possible I will say change everything and start fresh with cells, reagents and baculovirus and a P0 stock. It will surely work.
Please follow the proliferation of the cells. They have to stop growing after proper infection. Keep the cells below 2 x 10E6 all the time, when they are not infected yet. Be sure that you purified the bacmid from a true and pure transduced clone (white, large and Gm Res; not pale blue). That makes sure that you have a pure recombinant bacmid for the transfection. Is it a secreted or an intracellular protein. Kinetics of expression may be different.
Some proteins do not fold well. They can be detected in the cell debris after centrifugation of the extract.
Your expression seems to be low (WB, silver stain). In this case you have to do all the controles step by step to do each step in an optimal way to get reproducible results.
What about the turn-over of your protein? Is it stable or fast degraded?
Good luck
Are you using the same virus stock each time? They do go off with age. If we notice this, we regenerate a fresh bacmid from the Fastbac vector and start over. This usually works.
Alternatively, are you harvesting at the same point? Maybe your protein hasn't accumulated enough, if you harvest too early, or has degraded if you are harvesting too late. Try running a time course.
As a suggestion, consider the timing of harvesting, as Richard suggests. Lysis of the insects is a major problem in insect cells (there is essentialy a gene v-cath in the bacmid- which encodes a viral protease that can be quite active espicially after cell lysis, apart from several proteases) which leads to degradation of product often. This can lead to your band not showing up or showing up at an odd size-spot. Consider harvesting your product no later than 48 hours after division stopped.
I am experiencing similar things as described above.I transfected Sf9 cells with PCR-verified bacmid and saw all the signs of infections during transfection and baculovirus amplication of P1 and P2,but I could not see the specific band with Western and SDS-PAGE for P2 and P3 and P3 virus-infected(with varying MOI and time) cell lysates,can anyone give me some suggestion?Thanks.The Bac-to-Bac manual says"Viral stock contains a mixture of recombinant and non-recombinant baculovirus",however,the PCR of purified Bacmid gives only a specific band of expected size,without a 300bp band.The cells are low-passage healthy ones.
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