发信人: sunnyday (艳阳天), 信区: Biology
标 题: Re: 请教一个大肠杆菌里面表达蛋白的基本问题
发信站: BBS 未名空间站 (Sat Apr 12 22:11:37 2008)

It is actually very common for some unstable protein to precipitate in E.
coli into something called exclusion bodies. You need to discuss this with
your boss and see what you should do. (DON'T just do it by yourself)
To deal with inclusion bodies, several methods can be tried:
1. induce expression at low temperature like 17 oC with low concentration of
IPTG
2. co-express chaperon protein with your protein
3. extract your protein with 8M urea or 6M Guanidine, and then try to
recover
it by dialysis
4. In vitro expression

Anyway, none of the above methods could guarantee to work, you need to
discuss
with your boss to see what option you should take

【 在 oop (Michael Jordan) 的大作中提到: 】
: 大侠，我做一堆mutant就是想让蛋白变得很不稳定，这样在ecoli里面就沉淀的还没见
: 过。但是如果是现在的情况，我就担心能不能purify出来了。我会试试降低温度表达的
: 。现在是25度，下次试试20度。
: 不过上triton的话，还能弄掉去做CD或者NMR吗？谢谢！



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