Insoluble GST fusion protein help

Nina Baltes chaotropic at gmx.de
Wed Feb 18 11:43:45 EST 2004


Michael O'Hare wrote...
> Hi.  I need help purifying a highly insoluble GST-fusion protein.  I 
> don't neen lots of it, just enough to serve as substrate in a kinase 
> assay.  When I test my extracts from IPTG-induced vs non-induced I don't 
> see any differences in the banding pattern.   Any advice would be 
> appreciated.

If by insoluble you mean that the fusion protein forms aggregates in the 
cells, here's a protocol that isolates these:

- Induce a fresh 50 ml culture at OD660 = 0,3 - 0,5 with 1 mM IPTG for 2 
hours
- Centrifuge bacteria at 5000 rpm for 10 min 
- Resuspend cell pellet in 1-5 ml in 50 mM Tris buffer pH 8.0 with 25% 
sucrose 
- Freeze at -70°C for 30 min or over night 
- Thaw, then add 0,25 ml lysozyme (10 mg/ml in 250 mM Tris buffer, pH 
8.0) put on ice for 10 min.
- Add 10 ml 2x RIPA/TET (5:4, see below), put on ice for 5-10 min
- sonicate: duty cycle 50%; timer 1 min, full power; until solution is 
thin and opalescent; don't let it warm up!
- Centrifuge for 20 min at 15.000 rpm 
- resuspend pellet in 500 µl A. dest.

The pellet should be clear, sometimes there's also some strange 
insoluble black material in it, but this could be lab-specific ;).

2X RIPA 

20 mM 		Tris, pH 7.4
300 mM 	NaCl
2% 		Na-Deoxycholic Salt
2% 		NP-40 (Tertigol)

TET 

100 mM 	Tris, pH 8.0
50 mM 		EDTA, pH 8.0
2% 		Triton-X 100

1 µl of the resuspended aggregate usually gives a fat band on an SDS 
minigel, with little "background". However, it's quite insoluble (duh), 
so you'd probably have to experiment with different resuspension buffers 
or treatments to standardize the amount for your enzyme assay (I've 
never tried this though, so I can't help you there ;)).

Good luck!

Nina
-- 
C'est les microbes qui auront le dernier mot.
                            Louis Pasteur
http://www.chaotropic.net



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