inclusions from pET vector expresion

hraaxl at marc.cri.nz hraaxl at marc.cri.nz
Tue May 9 10:58:58 EST 1995


Dear protein expression in bugs sufferers

I'm after ideas. We have a viral non-structural protein expressed nicely using 
a pET vector. Problem is we get quite variable lysis of the bugs. I'll give 
some details of the way we do the expression and if anyone has improvements 
I'd love to hear about it.

1   we use 200 ml LB cultures. We use pGP-1 and heat induction to induce 
expression of the pET vector.
2   So, 1/40 dilution of overnight in LB, grow at 28 oC, 3 h
3   induce at 42 oC 1 h 
4   grow 37 oC 3 h
5   pellet bugs
6   resuspend in 50 ml: 50mM Tris, pH 8, 1 mM edta, 0.1 M NaCl, 0.1 mM PMSF, 
200 ug/ml lysozyme, 20 ug/ml DNAase, 20 ug/ml RNAase. Incubate room temp 30 
min to 3 h.
7   sonicate
8   pellet and resuspend in 5 ml wash buffer: 50 mM Tris pH 8, 0.1 mM NaCl, 10 
mM EDTA, 1 % triton x100
9   sonicate again, pellet and wash once or twice more


Problem is that after all this a huge gluggy mess of bug bits makes getting at 
the inclusions impossible/difficult.

Anyone do freeze/thaw to crack open the cells? Any good? How do you get 
solubilised bugs or smash them up so small they are no longer a problem? Are 
we trying to play with too great a volume of bugs?

Cheers

Tony Lough
HortResearch
Mt Albert Research Centre
Auckland, NZ



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