Hi! I have trouble finding literature on this. Can someone please share some information on what could be the cause of a glycoprotein-Fc-tag fusion protein being expressed in HEK cells missing its Fc tag? A smaller fraction of it (less than half) has the tag and seems to be the proper product I am looking for, but the larger half of the product is just the protein with the Fc tag missing. SDS Page and anti-Fc Western blot can't see any separate Fc tags. Can anyone point me to some literature or have some explanations from experience about what can cause this and what to do to fix it?
Hi Akos
it is not so clear your message. Usually you can find some information in the manual of the vector you are using. How do you detect your protein without tag?
Loosing the tag is possible but ofter the epitop is hidden in the protein. Some vectors have cleavage sites to obtain free tag proteins
Based in my experience there are other possibilities
1) the protein is digested by some protease during synthesis or during purification
2) the shorter protein is an intermediate product of synthesis, i.e. the messengers have some rare codons that block the translation
3) A second ATG exists in the messenger which gives rise to a protein in another frame which subtracts messenger to the syntesis of the right protein
Anyway all these events are rare ...
Paola Di Bonito Thanks for the answer!
I also have an antibody that is specific for the target protein I am expressing, so I can make a western blot for both the target and the Fc tag seperately.
A large part of the product is the size of just the normal recombinant protein without the tag, and reacts with the anti target protein antibody, but not the anti-Fc antibody. Another part of it is the proper size for the target+Fctag, and reacts with both antibodies.
I would need to purify my product using the FC tag on a ProtG column, so the part of the product that doesn't carry the Fc tag is lost to me.
I would like to find out why such a large amount of the product doesn't carry the Fc tag, and what could be causing this, so I can increase my yields.
Hi there,
I think proteolysis would be the simplest explanation. Is the protein secreted or located in intracellular vesicles?
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