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 > 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
