Dear Jin

are you sure that degradation happen in the linker? did you check it by n term sequencing or it is just an estimation from mw in sds page?

if the fragmentation is due to protease and happen during the culture you can try to decrease the induction temperature to see if a lower temperature decrease the protease activity and your degradation is reduced.

orherwise you can try to reduce you linker lenght ( eg use the gsgggg) or replace it with a non flexible linker (es EAAAK)n linkers.

just 2 quiestions:

1)are your 3 proteins very similar one with the other? 2) is it possible the the n- or the c- term of one of the 2 proteins linked is flexible and non structured ( eg predicted as coil) and digestion occour there and not in the linker?

good luck.

Manuele

proteases?

1. It is just an estimation from the sds page

2. The proteins are expressed in 18 degrees C

3. of course shorter linkers or other linker types can be used... but even so, the breaking(?) of linkers seem weird

4. the three proteins are all different

5. the three proteins are also structured