GST fusion prot.- HELP

aduarte at vax.oxford.ac.uk aduarte at vax.oxford.ac.uk
Mon Nov 7 09:09:13 EST 1994


Hello,

I am currently trying to express GST fusions proteins  (Approx. 55 KDa) 
using the pGEX expression vector. These proteins are purified by affinitty 
chromatography with a glutathione collumn and are used in DNAse I footprinting 
assays.

The level of expression is good and I consistently collect high levels of
protein but there is considerable degradation, with the full-size protein
representing only about 50% of the purified extract with various other smaller
products. This causes me problems both when trying to quantify the protein and
in terms of validating the DNAse footprinting data, because of the
possibility that the degradation products bind to the DNA target sequences with
modified specificity.

I suspect the degradation occurs while still growing the cells because I use
protease inhibitors (aprotinin, leupeptin and pepstatin, 3ug/ml each) and
apply a brief sonication treatment while keeping the cells on ice all the time
and working in the cold room.

I have tried changing the bacterial strain and used a protease deficient (lon-
and ompT-) strain from Novagen (BLR) without success. It seems that the strain
used may have an unpredictable effect in the stability of the protein being
expressed and I would like to test other strains. I also tried to reduce
degradation by adding the IPTG later in the course of the culture and keeping
the induction period to a minimum.

Can you suggest me potential good strains for protein expression or any tips
that may help to reduce the protein degradation (I'll enclose a summary of my
protocol)? 


Thank you very much.

Antonio Duarte (aduarte at vax.ox.ac.uk)
CRC Growth Factors
Dept. Zoology
Oxford



PROTOCOL

(1) Inoculate 1 ml of an overnight culture into 100 ml of LB-Ampicillin 
(50 ug/ml) medium. Grow the cells until OD550=1.0 and induce with 0.1 mM IPTG.
Incubate for 4 hours.

(2) Harvest the cells by centrifuging for 10 min at 3000 rpm
(4 C). Ressuspend in 1 ml of ice cold PBS, transfer to a 1.5 ml Eppendorf tube,
add protease inhibitors (as described above). 

(3) Sonicate for 15 secs with a 0.5 cm (diameter) probe. 
Add triton X 100 to 1% and centrifuge for 5 min in a microfuge. 

(4) Collect supernatant and add 200 ul of 50% glutathione agarose beads
slurry. Incubate for 20 min in a rotating wheel at 4 C. 

(5) Wash the beads several times with ice cold PBS and elute the 
fusion proteins with a 20 mM glutathion, 50 mM Tris pH 8.0 solution, 
containing protease inhibitors.

(6) Evaluate the protein produts on a Coomassie stained SDS/PAGE gel



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