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 <[log in to unmask]> 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
> [log in to unmask]
> [log in to unmask]
>
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