removing sarkosyl after lysis

P.J. van Santbrink santbrin at CHEM.LEIDENUNIV.NL
Wed Jun 25 03:47:23 EST 1997


Dear fellow netters,

I'm trying to isolate different GST-fusion proteins from E. coli (strain
AD 494 from Novagen) using the method published by Frangioni et al
(lysis with sarkosyl, isolation with glutathione 4B sepharose beads
AFTER the addition of Triton X-100 etc.).

Though the lysis with sarkosyl sees to it that about 50 % of my fusion
protein gets
into the supernatant (which is good) I'm not able to bind the protein to
the beads (which is NOT good).
I've tried different amounts of Triton X-100 (up to 5%; I lyse the cells
with 2% sarkosyl), different incubation times and I tried dialysis (for
up to 6 hours, 40C).

The sequence of my different fusion proteins has been checked.
The size of the different fusion proteins run from 36 kD to 63 kD (the
do have a lot of cysteine residues, that's why I used the strain AD 494
which allows the formation of disulfide bridges in the cytoplasm).

Question: Does anybody had similar problems (and did you find a
solution)? 
Is there an other way to get rid of the sarkosyl after lysis because I'm
pretty sure it's the presence of the sarkosyl that's preventing the
binding (control - empty vector pGEX - lysed without sarkosyl, binds
good to the beads).

I hope somebody can help me because I've run out of options !


Peter van santbrink
Division of Biopharmaceutics
University of Leiden (The Netherlands)


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