Hi,
I recently used an old P0 Baculovirus (stored at 4C for 8-9 months) to produce a viral glycoprotein (37kd). My previous expression for this protein using SF9 cells was always very low so I thought of increasing titer of old P0 virus and use Hi5 cells for expression. To increase the titer, I inoculated a monolayer of SF9 cells in a T25 flask with 200 microliter of old P0 stock and incubated at 28C for 5 days. Supernatant was harvested on day 5 and used (0.5ml) to produce P1 virus (50ml) using SF9 cells. Titer of P1 virus by plaque assay was 1 x 108 PFU/ml. Then I inoculated a 20ml suspension culture of Hi5 cells (1x106 cells/ml) with P1 virus at an MOI of 5 and incubated at 28C. 1ml cells were harvested at 48h, 72h and 96 h. Western blot revealed multiple bands around my target protein (37kd) for both P1 virus cell lysate (prepared in SF9 cells) and Hi5 cell lysates (at each time point). See image. I always use HALT protease inhibitor cocktail in my lysis buffer and do all steps on ice. Never had this problem before. I then tried to purify the protein using TALON and no protein was detected on western blot using His-tag primary and secondary antibodies. The original P0 virus stock is around 8-9 months old and stored at 4C (no FBS). Few months back, P1 and P2 virus generated from this P0 stock worked fine and produced only a single band of 37kd. But expression was very low. Should I start new transfection?
Thanks
Deepak
Yes, you can start a new batch and make a fresh virus, however, it is hard to blame the virus for this. Another possibility is because of the way you treated protein - it is degraded, and you don't have anything from the column. It is always better to have everything fresh, make a good titer, and then figure out more about the yield.
you state that your protein is a glycoprotein, so perhaps you are seeing different states of glycosylation.
Thanks Mahesh. I do not think the protein is degraded. I repeated the same experiment but this time also included an old pellet to see if my processing was OK. I could see a single band on western blot for the old pellet but not for the new ones. I am not able to get any protein from these lysates. I use TALON and it has worked fine before when I had a single band of protein. Now the protein is coming in the Flow through.Not binding to the column at all. I think these multiple bands are due to glycosylation. Do you think using Urea or Guanidine will make the tag available for binding?
Thanks Michael. I also think these multiple bands are due to glycosylation because they are close to the target protein (37kd). Problem is, I am not able to get any protein from these lysates. I use TALON , which worked fine before when I had a single band of protein. Protein is now coming in the flow through. Do you have any suggestion for bnding of glycosylated proteins to the column? I am now thinking to use denaturing conditions for purification.
If your protein is happy, stick to pH 8. Yes, the glycosylated protein will show multiple bands, but your protein shows one band at one time, and four-band at another time is somewhat confusing. Talon can work for one protein, but for some proteins it may not work. For denaturation, divide protein solution in equal half- add Gdn in one and keep others as control and load them to the same amount of resin. In parallel, make new prep of virus - Many things will fall in line if the yield is incredible.
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