Dear All, we are purifying a small secreted protein from conditioned media and have a rather unusual problem.
It is a small ectodomain (~11 kDa, pI ~6, His-tagged) of the type 1 transmembrane receptor, crystal structures are known (of the protein that was produced in E.coli and refolded; we are secreting the same protein using mammalian cells) so we can design reasonable constructs. The protein is expressed and secreted by transiently transfected HEK293T cells that work very well for other ectodomains and extracellular proteins in our hands (PMID 17001101). The target protein has 10 cysteines that form 5 disulfides in the crystal structure (of E.coli-expressed and refolded protein), there should be no free cysteines and no non-specific disulfides. Unfortunately, once the protein is secreted, it forms non-specific dimers and higher-order oligomers in the media (standard DMEM/2% FBS) before purification (confirmed by Western blotting under non-reducing conditions). Using 0.5 mM DTT during SEC gives a nice monomeric peak (however, the protein suffers as suggested by weaker interactions with its binding partners). We don't understand how a secreted protein (which passes trafficking quality control in the cell) with a known disulfide pattern forms non-specific disulfide linked oligomers in the extracellular media. We tried expressing it at 37 C and 30 C, and have sequenced our constructs (plasmids) multiple times. If anyone has seen this kind of problem and successfully solved it (purified homogeneous crystallisation quality protein), please let us know if possible. I thank you for your help. Best wishes, Tomas Dr. Tomas Malinauskas University of Oxford Wellcome Centre for Human Genetics Division of Structural Biology Roosevelt Drive Oxford OX3 7BN United Kingdom to...@strubi.ox.ac.uk tomas.malinaus...@gmail.com ######################################################################## To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB&A=1
