In article <5c7s0p$2du at bioalp.biobase.dk>, wind at biobase.dk (Troels Wind) wrote:
> Dear Netters,
>> I'm currently expressing a protein that is translocated to the periplasmic
> space by the Pel-B leader sequence. I have experienced some difficulties
> purifying the periplasmic space, thus I ask if any of you have some advice.
>> So far I have tried various osmotic shock strategies, but they do not seem
> to be very efficient, since most of the processed protein (i.e. without the
> Pel-B leader) is in the 'cytosolic' fraction obtained by freeze-thaw.
>> Has any of you tried a combination of lysozyme treatment AND osmotic shock?
> If so, would you tell me how? I tried lysozyme alone, but it wasnt very
> efficient either.
>> Thanks in advance for any input,
>> Troels Wind, M.Sc.
> University of Aarhus
Pharmacia suggests the following in their phage display system for the
extraction of periplasmic produced proteins:
resuspend pellet (equivalent of 5 ml coli-culture) in 0.5 ml icecold 1 x TES
add 0.75 ml icecold 0.2 x TES, vortex
on ice, 30 min, transfer in eppi
centrifuge, max, 10 min, 4°C
store supernatant at -20°C
TES-buffer: 0.2 M Tris/HCl (pH 8.0), 0.5 mM EDTA; 0.5 M Sucrose, sterilfiltrate
What do you mean by saying "the 'cytosolic' fraction obtained by
freeze-thaw". How do you know its cytosolic and not periplasmic protein.