p.j. van santbrink
santbrin at CHEM.LEIDENUNIV.NL
Tue Nov 12 11:10:08 EST 1996
I've read that by using betaine and sorbitol in
your medium you have a better change to get a
fusion protein in the soluble fraction after lysis
of your bacteria (and not in inclusion bodies).
I'am using the pGEX system of Pharmacia and in
pilot experiments I indeed managed to get both
GST and GST+protein in the soluble fraction after
But when I tried to get other fusion proteins in
the soluble fraction by the same method I didn't
succeed (almost everything in the insoluble fraction).
Those other fusion proteins are very similar to the
one I did get in the soluble fraction so I'm really
Could somebody tell me more about this betaine/sorbitol
method and tell me the essential steps in the procedure
(f.i. how important is the OD600 at which you induce your bugs).
Protocol I use, short version (send E-mail for detailed
protocol if you need it to give an answer to my problem):
- plate bugs from -80 stock (not thawed) on LB-agar plate
suplemented with amp, 2,5 mM Betaine and 0,1 M Sorbitol
- pick free colony from plate and grow (overnight)in
LB + the above mentioned suplements.
- dilute O/N culture 1/10 in fresh medium and incubate
1,5 hour 37 degrees Celsius.
- put bugs at room temperature and induce with IPTG (final
concentration 0,5 mM): incubate at 30 degrees Celsius,
225 rpm for 2 hours.
- lyse bugs with lysozyme (1 mg/ml) and 0,2% triton X-100
- after lysis centrifuge (14 k, 10 minutes): supernatant is
soluble fraction, pellet is insoluble fraction).
- bring part of fractions on SDS PAGE gel to look at results.
I hope somebody can help me.
Peter van Santbrink
University of Leiden
E-mail: santbrin at lacdr.LeidenUniv.nl
"Is a dream a lie if it don't come true
or is it something worse ?"
Bruce Springsteen, The River
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