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Thanks! We will try a DNA dilution trick. Yes, a fraction (~30-70%) is a monomer we want. On Fri, Dec 14, 2018 at 6:12 PM <[log in to unmask]> wrote: > > Hi Tomas, > > I have seen something similar in the past, see PMID: 26998761. The problem > could be alleviated by tuning down expression levels, through plasmid > dilution. I guess that cellular QC mechanisms are overwhelmed if you push too > hard for overexpression. I'd test a range of 1:10 to 1:1000, or higher, > dilutions in empty (pLEXm or any irrelevant plasmid) to keep total DNA amounts > constant. Is there no hint of monomer whatsoever in your prep? > > Best wishes, > > Radu > > -- > Radu Aricescu > MRC Laboratory of Molecular Biology > Francis Crick Avenue > Cambridge Biomedical Campus > Cambridge CB2 0QH, U.K. > tel: +44-(0)1223-267049 > fax: +44-(0)1223-268305 > www: http://www2.mrc-lmb.cam.ac.uk/group-leaders/a-to-g/radu-aricescu > > > 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 > > [log in to unmask] > > [log in to unmask] > > > > ######################################################################## > > > > 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