Dear Dima,
thank you for your reply
Since this is ectodomain, I presume it contains native sorting sequence.
The ectodomain contains a secretion signal: either its native or
another secretion signal. I have tested a few other, strong secretion
signals to boost the expression levels with moderate success. A
reference on the use of different secretion signals is Barash et al.,
Biochem Biophys Res Commun. 2002 Jun 21;294(4):835-42.
In which case you don't have to worry about disulfide formation -
your protein will pass through golgi which is oxidative environment
favoring S-S bridges.
Protein folding and disulfide formation happens in the oxidative
environment of the ER. PDI is ER-resident.
(The PDI thing you mention probably refers to
E.coli; I've never heard about it in mammalian overexpressions).
No, I do not refer to the E.coli PDI. Mammalian PDI has been
overexpressed in mammalian cells to enhance the expression level of
secreted recombinant protein.
e.g. human PDI in Davis et al., Biotechnol Prog. 2000 Sep-Oct;16(5):736-43.
With regard to low levels of expression: Is there a particular reason
to use 293 cells? Transient expression in Cos-1/Cos-7 cells gives
~ 10 fold higher levels of expression because of the plasmid amplification
in these cells. (The only requirement is that the plasmid should
have SV40 ori; most mammalian vectors do).
HEK293T cells are reported to have high expression levels, especially
for secretted proteins (Aricescu et al., ACTA D, 2006).
Dear all,
sorry for this off topic question. I am producing the ectodomain of a
human receptor in
HEK293T cells by transient expression. The expression levels are quite
low. The protein
contains a high number of disulfide bridges. It has been reported that
overexpression of
protein disulfide isomerase can enhance the secretion of proteins with
a high disulfide
content. I would like to test the effect of PDI by cotransfecting a
PDI-encoding vector
with my expression plasmid. I wonder if anybody has some experience with
PDI-overexpression and if anybody could provide me an mammalian
expression construct for
PDI?
Any suggestions are appreciated,
Kenneth Verstraete
Unit for Structural Biology and Biophysics
Laboratory for Protein Biochemistry and Biomolecular Engineering
Ghent University
K.L. Ledeganckstraat 35
9000 Ghent, BELGIUM
Phone: +32-(0)9-264.51.26
<http://www.eiwitbiochemie.ugent.be/units_en/structbio_en.html>
