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
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