I expressed one protein in insect cell using bac-to-bac system. The protein was secreted in 293T cell. But it was stucked in cytoplasm. The protein was aggregated when it was lysised with different lysis buffer even resuspension with high concentration salt, 2-ME and Urea. It only precipitated with 5000g for 20 min. It could not be loaded to the column. How can I solve this problem?
Hi Yi Wang,
I'm a bit confused as to what you have done here.
Have you tried to express a baculovirus in 293T cells? 293T cells are mammalian and you will not get expression in them from a standard baculovirus.
Or are you saying that a protein that expressed and secreted in 293T cells doesn't do so when expressed in insect cells? If so, which insect cells? High5s sometimes give secretion when Sf9s or Sf21s don't.
As for lysis buffer, this is really a protein question and could require screening of a range of buffers, pH and salt conditions. However if the protein is already insoluble in the cells the buffer conditions will not help make it soluble short of going down a refolding route.
More information as to what you have done will help.
-Richard
Thank you answer! the protein could be expressed and secreted in 293T cell. When I transfered the expression to sf-9 cell, I found the protien it could not be secreted without transmembrane domain. I tried several lysis buffer and still could not get the soluble protein. As you had said, it maybe already insoluble in the cells.
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