Hi Tomas, In addition to all other suggestions, and as Zhijie suggests, a good alternative strategy to plasmid dilution may be inducible expression using the HEK293S GnTI– TetR cell line (PMID: 12370423) and a compatible expression vector such as pACMV-TetO (PMID: 22322218) or pHR-CMV-TetO2 (PMID: 30455477, sorry for the shameless self-advertising ;) ). In principle these plasmids can be used for transient expression as well if you do not want to devote time to establishment of mono- or polyclonal cell lines.
My educated guess is that titration of the TetR-TetO system with Doxycycline yields similar effects to plasmid dilution; more controlled and less overwhelming transcription, translation, and passage through the ER and secretory pathway; with lower overall expression levels but higher secreted levels as a result. Cheers, Jonathan E. > On 14 Dec 2018, at 17:55, Tomas Malinauskas <[email protected]> > 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 > [email protected] > [email protected] > > ######################################################################## > > 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
