problems with cleaving fusion protein

Artem Evdokimov AEVDOKIMOZ at cinci.rr.com
Wed Apr 10 21:44:11 EST 2002


Hi,

Unfortunately, sometimes the linker between fusion proteins becomes
inaccessible - many reasons are possible but there is no clear way to solve
the problem. Stuff that worked for us in the past:

1) increase the length of the linker using poly-Gly or
Pro-Gly-Gly-Gly-Gly-Pro. Make sure that your cut is after the linker so that
your final product isn't suffering (unless a few extra residues won't hurt
what you're going to do with the final product).
2) change pH. Screen cleavage efficiency vs pH.
3) change proteases (TEV is our favorite)
4) make sure that the MBP is loaded with maltose (yes, sometimes it
matters!) or if you tried loaded first, try to avoid loading.
5) If the above fails, consider increasing linker length on the side of the
target fusion protein - you're taking a chance that a few extra residues
won't matter.

Good luck, let us know if anything worked.

A.G.E.

"Iva Toudjarska" <toudjarska at wi.mit.edu> wrote in message
news:3CB47BFC.6A80A2AF at wi.mit.edu...
> 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
>
>
>





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