Oliver, first, what is the source organism of the protein, human, dog, cat, insect, or bacterial? second, how big is the native protein, and how big are the tags you've added? third, how many transmembrane spans are predicted (use any of the million programs available) fourth, when you seperate the soluble from the insoluble protein, do it in three steps. Isolate the aqueous/salt (500mM) portion, vs a 8M urea wash fraction vs, a final detergent soluble fraction. Do Ni chromatography, CCB stain it, and send a picture of it. Then we can have a better understanding of what is what. Paul
Dr. Paul Kraft Structural Biologist cell 586-596-2770 email: [email protected] The information constituting this transmission is privileged and confidential. If you are not the intended recipient, please notify me immediately. --- On Tue, 6/8/10, Oliver Li <[email protected]> wrote: From: Oliver Li <[email protected]> Subject: [ccp4bb] purification of a strange protein To: [email protected] Date: Tuesday, June 8, 2010, 2:13 AM 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
