I worked for several years on mammalian cell expression of extra-cellular domains of single spanning membrane proteins and other membrane proteins. Just like you did when we switched stable lines from serum containing to serum free medium the expression drastically reduced.
So after switching to serum free medium did you try picking clones in selective medium again and try to look for adapted clones to the new medium? No protocol suggest this but i had good luck. I am not very sure this is exactly your problem if so what you see is not very unusual. I do not understadn why you switch from your CHO stable line expression? In my opinion stick with stable clones in serum containing medium or serum free medium (not really necessary since it will run very expensive in the long run) since you are using affinity tags to pull your protein of interest from secreted protein in the medium a batch binding of your media in a roller bottle apparatus over several hours will help solve your binding issues. Identify ideal washes to get rid of serum binding or reduce non-specific binding. I am surprised how hyper-glycosylation was not a problem when HEK is used. To save a bunch of money on deglycosylation enzymes you should switch to LEC mutants of CHO cell lines. Padayatti On Mon, May 19, 2014 at 7:13 AM, Bernhard Rupp <hofkristall...@gmail.com>wrote: > Hi Fellows, > > > > my lab mates successfully expressed a glycoprotein in CHO cells in serum > free medium, and > > the protein captures nicely on HisTrap Excel 1ml columns (obviously, high > yield is not my problem…). > > We load ca 1L supernatant at 0.5 ml/min, and eluate with a steep imidazole > gradient. 20mM Imidazole buffer for regeneration. > > Works fine (and often…see yield remark). > > > > Overcome by common crystallographers’ greed (nor creed), we switched to > stable xfected HEK293, and cell free medium Gibco CD 293. > > The first run gave high final yields & cheers. > > The second run less of either, because the small HisTrap column > essentially dissolved – the medium collapsed, > > Ni leaches out, kaput as kaput goes. > > A 3rd run on a similar previously working column lead to the same result. > > > > Only thing changed was the cells and medium. Same buffers, same gradients, > same Akta equipment, same lab techs. > > > > Before I improve the statistics by ruining further columns, has anybody > experienced such a calamity that might > > be blamable on secret media components or similar? There is a mysterious > ‘proprietary dispersant’ preventing > > cell adhesion quoted…. > > > > Best wishes, BR > > > > > ---------------------------------------------------------------------------------------- > > Bernhard Rupp > > b...@ruppweb.org > > b...@hofkristallamt.org > > http://www.ruppweb.org/ > > ----------------------------------------------------------------------- > > > > > -- P
