Dear Zhijie et al, thanks a lot for your thoughtful suggestions. a) we do get a fraction with a "proper" monomer but it would be nice to minimise losses because of non-specific disulfides; b) yes, it has 1 Asn-linked glycan which gets nicely trimmed upon treatment with Endo F1; d) yes, we do get some monomer on SEC without DTT. We thought ~0.5 mM DTT (which degraded relatively fast anyway) might be OK to reduce some non-specific disulfides but not sufficiently high to destroy the whole folded domain, we used it out of desperation/curiosity; e) we will try longer tags and adding more residues at termini. Best wishes, Tomas
On Fri, Dec 14, 2018 at 6:57 PM Zhijie Li <zhijie...@utoronto.ca> wrote: > > 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
