s******y
发帖数: 28562 | |
C*********m
发帖数: 213 | 2 leucine zipper?
which
these
any
【在 s******y 的大作中提到】
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s******y
发帖数: 28562 | 3 That is possible, but can you suggest a particular leucine zipper (name or
sequence) in particular?
I feel embarassed because I don't remember anyone of them right now.
【在 C*********m 的大作中提到】
: leucine zipper?
:
: which
: these
: any
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l****y
发帖数: 398 | 4 GCN4
more controllable-fkbp/frb
【在 s******y 的大作中提到】
: That is possible, but can you suggest a particular leucine zipper (name or
: sequence) in particular?
: I feel embarassed because I don't remember anyone of them right now.
|
s******y
发帖数: 28562 | 5 Wow, the fkbp/frb seems like a cool stuff with super high affinity!
I am definitly going to read a lot more of the literature about it. The only concern I have is those domains are bigger than my expectation.
The GCN leucine zipper sounds like a good idea because it is smaller, but the affinity is so so...let me see if they have mutants with higher affinity...
【在 l****y 的大作中提到】
: GCN4
: more controllable-fkbp/frb
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w******e
发帖数: 1187 | 6 conjugate to ssDNA/RNA using like NHS chemistry?
which
these
any
【在 s******y 的大作中提到】
: Wow, the fkbp/frb seems like a cool stuff with super high affinity!
: I am definitly going to read a lot more of the literature about it. The only concern I have is those domains are bigger than my expectation.
: The GCN leucine zipper sounds like a good idea because it is smaller, but the affinity is so so...let me see if they have mutants with higher affinity...
|
s******y
发帖数: 28562 | 7 Unfortunately we cannot do that either because we need to express the
protein in vivo.
Conjugate them with nucleotide can only be done in vitro and there is no
guarantee where the connection site is.
【在 w******e 的大作中提到】
: conjugate to ssDNA/RNA using like NHS chemistry?
:
: which
: these
: any
|
y******8
发帖数: 1764 | 8 how about Protein G B1 domain and binding motif on Fc |
y******8
发帖数: 1764 | 9 Don't know the size limits. If the fusion domain can go above 25kd, then GST
is a very good choice. |
s******y
发帖数: 28562 | 10 这个应该也可以。但是我担心protein A or G 的binding domain 有人
单独用过么？就是不包含其他序列，光用那个binding domain，能和Fc 里面的
单独的motif结合么？
GST蛋白不行。太大，而且GST 会导致转录出来的蛋白直接就成了homodimer 了。
其实，我刚才想了一下，发现不能用dimerization domain, 因为一旦成了
homodimer 就麻烦了，最好还是heterodimer, 象你说的那个Protein G + Fc domain
【在 y******8 的大作中提到】
: how about Protein G B1 domain and binding motif on Fc
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|
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y******8
发帖数: 1764 | 11 Tandem repeats of motif could increase the affinity by 10 fold or even more.
Homodimerization could be diminished by polycistronic expression.
By the way, if you are willing to dig the bacterial protein database, there
are many many possibilities. In general, those proteins are small and
readily folded. The affinity of those interaction are tremendously high.
【在 s******y 的大作中提到】
: 这个应该也可以。但是我担心protein A or G 的binding domain 有人
: 单独用过么？就是不包含其他序列，光用那个binding domain，能和Fc 里面的
: 单独的motif结合么？
: GST蛋白不行。太大，而且GST 会导致转录出来的蛋白直接就成了homodimer 了。
: 其实，我刚才想了一下，发现不能用dimerization domain, 因为一旦成了
: homodimer 就麻烦了，最好还是heterodimer, 象你说的那个Protein G + Fc domain
|
y******8
发帖数: 1764 | 12 I think people hold patents on all of these. You might need to get approval
first.
【在 s******y 的大作中提到】
: 这个应该也可以。但是我担心protein A or G 的binding domain 有人
: 单独用过么？就是不包含其他序列，光用那个binding domain，能和Fc 里面的
: 单独的motif结合么？
: GST蛋白不行。太大，而且GST 会导致转录出来的蛋白直接就成了homodimer 了。
: 其实，我刚才想了一下，发现不能用dimerization domain, 因为一旦成了
: homodimer 就麻烦了，最好还是heterodimer, 象你说的那个Protein G + Fc domain
|
s******y
发帖数: 28562 | 13 唉，我就是不知道该到哪里去找这种数据库啊？
我不管是什么来源的蛋白，只要那个蛋白不会杀死表达的动物细胞就好了。
有什么建议怎么去查这种数据库？
more.
there
【在 y******8 的大作中提到】
: Tandem repeats of motif could increase the affinity by 10 fold or even more.
: Homodimerization could be diminished by polycistronic expression.
: By the way, if you are willing to dig the bacterial protein database, there
: are many many possibilities. In general, those proteins are small and
: readily folded. The affinity of those interaction are tremendously high.
|
s******y
发帖数: 28562 | 14 啊呀这么麻烦啊？难道这种类似的实验以前没有人设计过么？
approval
【在 y******8 的大作中提到】
: I think people hold patents on all of these. You might need to get approval
: first.
|
y******8
发帖数: 1764 | 15 The protein G B1 domain is about 8kd, and would be hard to synthesize.
Pratically, this is the only reason you need to ask for the material and
permission to start. Otherwise, you could just do it and wait for response.
Usually, people don't pay attention to nonprofit stuff.
【在 s******y 的大作中提到】
: 啊呀这么麻烦啊？难道这种类似的实验以前没有人设计过么？
:
: approval
|
y******8
发帖数: 1764 | 16 Neither do I. But very often, I am amazed by those discoveries from
microbiologists.
【在 s******y 的大作中提到】
: 唉，我就是不知道该到哪里去找这种数据库啊？
: 我不管是什么来源的蛋白，只要那个蛋白不会杀死表达的动物细胞就好了。
: 有什么建议怎么去查这种数据库？
:
: more.
: there
|
s******y
发帖数: 28562 | 17 OK... that could be quite a hassle then.
.
【在 y******8 的大作中提到】
: Neither do I. But very often, I am amazed by those discoveries from
: microbiologists.
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s******y
发帖数: 28562 | 18 再问一下，有没有人知道？
which
【在 s******y 的大作中提到】
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s******y
发帖数: 28562 | 19 小声地再问一句：
没有人知道么？
我其实要找的就是两个有比较高结合能力的蛋白domain，最好是长度都
不超过50个氨基酸的，而且序列已知的，而且是heterodimer.
which
【在 s******y 的大作中提到】
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d******1
发帖数: 709 | 20 Scaffold protein?
Like PDZ domain in neuron junction? two PDZ domains from two differnt
proteins pull them together to perform the function.
Zhang Mingjie in HKUST studied a lot on PDZ and PDZ heterdimerization. you
may check his website.
http://bcz102.ust.hk/index_publications.htm |
s******y
发帖数: 28562 | 21 谢谢。明天到实验室仔细看看去
【在 d******1 的大作中提到】
: Scaffold protein?
: Like PDZ domain in neuron junction? two PDZ domains from two differnt
: proteins pull them together to perform the function.
: Zhang Mingjie in HKUST studied a lot on PDZ and PDZ heterdimerization. you
: may check his website.
: http://bcz102.ust.hk/index_publications.htm
|
