Troubleshooting flag-tagged secreting protein expression - Troubleshooting, Improvement, tricks! (Sep/13/2005 )

My aim is to express and purify a flag-tag recombinant protein in cell line. This fusion protein has a natural signal peptide, so it will be secreted outside cell into culture medium evently. Flag tag locates after signal peptide, linked by a AG small linker next to signal peptide cleavage site. The coding sequence is inserted into pIRES2-EGFP. In addition, the small protein has a GPI anchor at its C terminal, so part of the protein will be membrane bounded.

I transfected the expression construct into 293T cell with lipofectamine 2000. The cell was then cultured in Opti-MEM with 1%FBS for 3days. Then I harvested the culture medium, removed cell debris, and had the peptide pull down using a M2 flag antibody(sigma) and Protein G. The product was then test by western blotting using Flag antibody.

While, I could only detect slight signal from IP product, and no signal could be detected in sample loaded only with supernatant, although high level of signal could be seen in cell lysate.

My question is:

How to improve the expression level?

Do I need to use an other signal peptide to improve secreting?

Do I need to cut membrane bounded protein with PI-PLC?

Dose stable transfectant(strain) has very higher expression level of target protein?

293T cell line is resistance to G418, which adhesion cell line is better for establishment of stable transfection?

Every comment is precious! You are welcome to give your advice!

Thank you very much!

first of all, what's your growing medium for 293T? DMEM 10%? i serum starve the cells in 1% FBS. dont know how exactly it looks with 293T, but after transfection i would simply put the growth medium on those cells to let them live and, most importantly, express, before you lyse them.

actually, I tried two culture medium. the first is DMEM + 10%FBS, while I was told that high serum ratio maked immunopricipitation difficult, so I replaced the medium with Opti-MEM plus only 1%FBS, and the 293T cell could grow with it for at least two days without obvious disruption of cell morphology, although cell proliferation was slowed down. One point should be noticed, that is my interested protein has a GPI anchor at its C-Terminal.
Thank you for your comments!

hmm.. i still would go up with serum. 293T need at least 3 -4 d without serum to start looking different. did you test if % of serum had an efect on your ip?
how big is your protein? can you tell what part of your protein binds to the membrane? and since you can detect the protein in the lysate, and not in culture supernatant, there is clearly some secretion problem. mystery for me. good luck anyway.

The secreted protein will be attatched on the cell membrane through a GPI anchor at its C terminal. It is only about 10kd. Because this protein can be regulated by activity, and its secretion pathway may be a regulative one, but not constitutive one, there may be some problem on secretion, just as you had mentioned. I put this protein after its own signal peptide, do you think that I should replace it with another one that can have strong effect on secretion?

Have you used pFlag-CMV-3plasmid for secretion protein expression in mammalian cell lines? How about its productivity?

THANKS A LOT!

hmm..  i still would go up with serum. 293T need at least 3 -4 d without serum to start looking different. did you test if % of serum had an efect on your ip?
how big is your protein? can you tell what part of your protein binds to the membrane? and since you can detect the protein in the lysate, and not in culture supernatant, there is clearly some secretion problem.  mystery for me. good luck anyway.

Ok. So I never worked with secretion protein, so dont know much about that.
But as I see it, your protein is pretty small, so in general you shouldnt have any problems if it goes for expression.
hmm.. do you think that putting FLAG after the signal peptide may somehow interfere with the cleavage? and therefore with secretion? you know, something like not proper folding or such a stuff.
the other thing is, that if your protein sticks to the membrane maybe, as you mentioned before, not much is floating around?
when you said, that you've seen expression in the lysate, is it possible that you detect the protein which is actually on the membrane, already secreted?
of course, replacing signal peptide for stronger would be not a bad idea, but first I would make sure it is necessary..

And for the expression, pFLAG-CMV3 - for me it expresses ok.
But pCG plasmids give more, although the same promoter