发信人: royluo ( 罗马情结), 信区: Biology
标 题: Re: questions on soluble expression
发信站: Unknown Space - 未名空间 (Tue May 17 10:47:10 2005) WWW-POST

I guess you are using a method called "Osmotic Shock" to release periplasm
protein from
the bacteria. But in this method, E.Coli is first resuspended in 20%sucrose
plus some EDTA,
then resuspended in cold water. I am not sure why you use MgSO4.
Your first question: 20% sucrose is used to increase the osmotic pressure
in the periplasm.
EDTA is to permeablize outer membrane of E.Coli. E.Coli is then resuspended in
cold water.
Because periplasm has a high osmotic pressure and the outer membrane have
holes now,
proteins in the periplasm will burst out into the cold water.
Frankly speaking, I hate this method. Because cytoplasmic membrane of
E.Coli is pretty fragile.
So, during the osmotic shock, some E.Coli are lysed already and the released
DNA makes the whole
solution very viscous. I did osmotic shock for a while and then gave it up. I
found out direct sonication
+Ni beads purification is very efficient already.


【 在 crystalmaker (风雨无阻) 的大作中提到: 】
: by using a leaser peptide, my protein is released in both LB and
periplasmid.
: According to the protocal, to release the protein from the cells, first use
: 20% sucrose, and then 5 mM MgSO4.
:
: 1. what is the purpose of 20% Sucrose? can I skip this step and use 5 mM
MgSO4
: directly?
:
: 2. I found in 20% Sucrose my protein is partly released, before using 5 mM
: MgSO4. Is this normal?
:
: 3. I feel ercovering the protein from LB is very time-consuming:
concentrating
: and dialysis, and then concentrate before loading to the column (Hitrapp
since
: my protein has a 6-His tag). Is there a simpler way?
:
: any comments are appreciated.
:


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