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
Dear Zhijie et al,
thanks a lot for your thoughtful suggestions.
a) we do get a fraction with a "proper" monomer but it would be nice
to minimise losses because of non-specific disulfides;
b) yes, it has 1 Asn-linked glycan which gets nicely trimmed upon
treatment with Endo F1;
d) yes, we do get
Dear Tomas,
Along Radu’s comments, here’s another paper well worth looking into:
Halff EF, Versteeg M, Brondijk TH, Huizinga EG
When less becomes more: optimization of protein expression in HEK293-EBNA1
cells using plasmid titration - a case study for NLRs
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,
Hi Tomas,
Some thoughts:
a) I guess the thermodynamic drive for all part of this small ectodomain to
fold into a single lowest energy conformation is not very strong. The cells
can’t know that the little artificial domain is supposed to be a monomer when
the oligomers are also sufficiently
Thanks! We will try a DNA dilution trick. Yes, a fraction (~30-70%) is
a monomer we want.
On Fri, Dec 14, 2018 at 6:12 PM 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
>
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
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
