Dear colleagues,
I am now trying to purify a strange protein. The known function of the
protein was little, and it was speculated that the protein linked on the
surface of cell membrane.
It was expressed with a Trx-tag at very low level of solubility.
Furthermore, the protein polymerized severely without detergent, and detergent
would make it existed as oligmer such as trimer.
If 1% Triton X-100 was added into the lysis buffer, almost all of the
protein existed in soluble manner. If the Trx-tag was cut off after Ni
chelating chromatography, the protein cannot be eluted from DEAE column even
the concentration of NaCl up to 1 M (the pI of the protein is about 5.6, the pH
of the eluting buffer is 8.0). While if the fusion protein after Ni chelating
chromatography was futher purified using DEAE or Q FF, most of fusion protein
cannot bind on the column even if the concentration of NaCl of the binding
buffer reduced to 0 M. Despite this, some fusion protein (probably one-quarter)
bond on the column and was eluted with the increase of ionic strength.
Unfortunately, the purity was poor. I wonder if this arose from the bad state
of the protein. The most peculiar thing perplexed me was that the protein
extracted by 1% Triton X-100 was inclusion body or Triton X-100 as a detergent
make the protein dissolve out.
Besides, I intend to regard it as a membrane protein during the process of
purification and crystallization, now.
Any suggestions about how to purify the protein will be appreciated!
Best wishes!
Oliver
