Yes, we have also seen some indication that overdriving the expression can cause problems. In our cases this may range from bad aggregations or loss of expression. Since we use induced stable cells we simply reduced the doxycyline levels.
Zhijie
> On Dec 14, 2018, at 1:13 PM, "[log in to unmask]" <[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]
>>
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