problems with cleaving fusion protein

Iva Toudjarska toudjarska at wi.mit.edu
Wed Apr 10 12:53:02 EST 2002


Hi guys,

I am expressing a part of Drosophila protein fusion with MBP. Originally

in pMAL vector there is Factor Xa protease cleavage site. I can express
the fusion protein at good levels with good soluble fraction, purify it
through amylose column. When I try to cleave this fusion with FXa at
concentrations 1, 2, 5 and 10 ug  protease per 100 ug fusion protein
cleavage rate is very very low even after 3 day of incubation on 4oC,
the rate was slightly higher at RT. Conditions were 20 mM Tris-HCl pH
7.4, 200 mM NaCl, 10 mM maltose, NO DTT(this is the elution buffer for
the amylose column, according to NEB FXa works in it). It did not get
better in presence of Urea. Some people said FXa is poor cleaver. So I
introduced site for PresCission protease (Amersham) verified the seq-
everything there, correct and ready to be cleaved. Protein was dialyzed
out against Cleavage Buffer. After 12 h incubation at 4oC with 1, 5, 10
ug PresCission Protease per 10 ug protein and 10 ug protease in presence

of 0.01% and 0.1% Tween 20 or Triton X100 I do not see a band
corresponding to my protein (21 KDa without MBP), I can't see MBP only
because of the presence of Prescision around the same size but the bend
does not appear to increase with time. I should mention that PresCision
protease got frozen at -20 oC. I am not sure if this affects the
activity. Now I'm waiting for a new tube from Amersham.

I will really appreciate some advise and if anyone can share experience
with these proteases or MBP fusion proteins
Theoretically based on the structure the linker between MBP and my
protein is supposed to be relaxed.
Please help!

Any input will be greatly appreciated

best regards Iva






More information about the Proteins mailing list