发信人: sunnyday (飞鸟), 信区: Biology
标 题: Re: 请教：GST fusion protein
发信站: The unknown SPACE (Sun Jan 19 22:09:51 2003) WWW-POST

In my own experience, GST is not a good tag because such reason:
1. the GST is on the C term, so , even the protein is not completely
expressed, the GST is still there.
2. GST has a tendency to form dimer (just like what others said)
In your case, it is very likely your protein wasn't expressed correctly.
You can try to induce it by a lower dose of IPTG. In our lab's experience,
sometime it helps to express big proteins. It is also a possibility that you
may introduce a stop codon inside your protein sequence. I suggest you to
sequence your vector again and make sure about it.
As to the small protein in mammalian, well, I have no idea if your vector is
alright.


【 在 Geneporter (Geneporter) 的大作中提到: 】
: I am struggling to express a GST fusion protein in E Coli (PGEX2T vector),
but
: everytime I got few short-sized protein expressed (but larger than GST)
looks
: like my protein (about 70-80 Kd with GST) is degraded, tried several times,
: could not get the big sized protein. At the same time, my small protein (45
: aas) fused with GST expressed very well and can detect strong band by
anti-GST
: western blot.
: Another problem is after I cloned these small GST fusion gene into mammalian
: vectors, then did transient transfection. I could not detect any anti-GST
band
: at all.
: Thanks for this puzzle, any suggestions will be highly appreciated.
:
:


--
※ 来源:．The unknown SPACE bbs.mit.edu．[FROM: 159.178.]