Hi Tomas, Some thoughts: a) I guess the thermodynamic drive for all part of this small ectodomain to fold into a single lowest energy conformation is not very strong. The cells can’t know that the little artificial domain is supposed to be a monomer when the oligomers are also sufficiently hydrophilic. We also occasionally see aggregates coming out of human cell lines. Sometimes barely anything good.
(One wild thought: the disulfide shuffling system in eukaryotic cells have something to do with the N-glycans. Does the natural form of this protein have N-glycans, while the ecotodomain is somehow deprived of that?) b) Is there absolutely no monomer on the Western? If this is the case, since the bacterial preps can be refolded, can you try incubating the protein with Cysteine or GSH or even add PDI or DsbC to try to increase the population of properly folded species? c) If you have some monomers or can manage to generate some by disulfide shuffling: for a small domain with such high disulfide content a further reverse-phase C18 HPLC step may give you the properly folded species without irreversibly denaturing them. Or maybe a Superdex75 gel filtration or a silica SEC on HPLC, without reducing reagents, see below. d) Have you ever run the gel filtration without DTT? When putting the DTT, because of its strong disulfide cleaving power, you might be sending the misfoled junk to the monomer peak. They look like monomers when DTT is present but they are misfolded still. If you have a small fraction of monomers that had passed the cell’s QC, they are more likely to be correctly folded therefore active. Then the right thing to do I think is to try to separate the monomers from the oligomers, rather than forcing everything into “monomers”. e) Try a larger tag? Add more natural sequence back to the domain? Try periplasmic expression in E coli? The NEB pMal-p vector is my favourite for disulfide forming proteins, when not using mammalian cells. Zhijie > On Dec 14, 2018, at 11:57 AM, Tomas Malinauskas <tomas.malinaus...@gmail.com> > wrote: > > 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 ######################################################################## To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB&A=1
