HER2/neu ECD Expression in CHO cells

Gregory P. Adams, Ph.D. gp_adams at fccc.edu
Thu Jun 17 01:43:14 EST 1999


Hi,

    We are looking for some advice on problems we are experiencing in
our expression of HER2/neu extracellular domain (ECD).  We are using the
pEE14.1 vector (Lonza) under control of the CMV promoter and glutamine
synthetase selection in CHO cells.  We have isolated the ECD gene by
RT-PCR and have inserted it along with the leader sequence, Kozak
sequence and ATG initiation site into the vector.  After transforming
the cells and growing them under selection conditions we have attempted
to isolate the ECD protein from both the supernatant and from cellular
lysates and have found that we get a protein that contains the
C-Terminal His-6 sequence and is reactive with some anti-HER2/neu MAbs
in western blots, but has a molecular weight approximately equal to an
unglycosylated form of the ECD (MW ~64 kDa instead of >100kDa).  We are
going to try to deglycosylate the protein and look for a shift in size
on the PAGE gel (the lack of which would indicate that the protein has
not been glycosylated as it was supposed to be).  At the present, we
suspect this to be the problem.  Does anyone have any suggestions as to
why this might be happening or suggestions of what else may be
occuring?  Thanks in advance for any advice!

-Greg
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