s******y
发帖数: 28562 | 1 我们现在需要设计一个linker system, 需要在两个蛋白之间放进一串小
蛋白来调整那两个蛋白的距离。要求长度可调（就是可以靠放进蛋白的数量来
调节linker 的长度）。我知道别人做类似的事情的时候会放进titin I27 domain,
但是现在的问题是我们要求这个可以调节的单位长度必须在 50 Aa以内，而
titin I27 domain正好比这个大一倍。
我不知道有什么蛋白的domain 是符合这个要求的？请大家推荐一下?就是必须能够
在５０Aa之内自发折叠成型（最好是球形），而且不要有太明显的功能区域（比方
说有个什么什么binding domain 就大大的不好）的东西。必须在５０个氨基酸内，
但也不要太短，6xHis 这种就太短了。２０～５０Aa最好。
谢谢！ |
c**i
发帖数: 265 | 2 这篇paper谈到了这个问题Design of linkers which effectively separate domains
of a bifunctional fusion protein
Protein A 的 一个3个alpha螺旋的domain可以做spacer，是55aa，略大于50。
他们也用了5个连续的alphahelix，30aa做spacer。 |
b******y
发帖数: 627 | 3 check "polyproline ruler" on pubmed. It might serve your purpose. |
s******y
发帖数: 28562 | 4 谢谢！！！
domains
【在 c**i 的大作中提到】
: 这篇paper谈到了这个问题Design of linkers which effectively separate domains
: of a bifunctional fusion protein
: Protein A 的 一个3个alpha螺旋的domain可以做spacer，是55aa，略大于50。
: 他们也用了5个连续的alphahelix，30aa做spacer。
|
s******y
发帖数: 28562 | 5 好的，我去看看先。
不过我对polyproline多少有点疑虑，因为polyproline 是一个已知的
binding domain for profilin.
【在 b******y 的大作中提到】
: check "polyproline ruler" on pubmed. It might serve your purpose.
|
b******y
发帖数: 627 | 6 yes, indeed.
does it has to be a folded domain? can it be some flexible linkers? |
b******y
发帖数: 627 | 7 roughly remember there was a science paper for designing highly stable
proteins by optimizing packing in the core. They were able to make a small
protein very stable. just don't remember which paper. if i recall, I will
let you know.
Make sure not to use ww domains (although they are small enough) because
they are all marginally stable and may form amyloid fiber if multiple ones
are used in a row. |
b******y
发帖数: 627 | 8 roughly remember there was a science paper for designing highly stable
proteins by optimizing packing in the core. They were able to make a small
protein very stable. just don't remember which paper. if i recall, I will
let you know.
Make sure not to use ww domains (although they are small enough) because
they are all marginally stable and may form amyloid fiber if multiple ones
are used in a row. |
s******9
发帖数: 283 | 9 E. coli ToA domain II, a very long alpha-helical domain and you can excise
any length of fragments from it for your purpose. It serves as a spacer
region for ribosome display. |
s******y
发帖数: 28562 | 10 我担心的是假如linker 是flexible 的话会干扰到两端的蛋白折叠。
我的想法是如果linker 本身有很好的折叠的话应该会自己折起来就不会折到其他地方
去了。
【在 b******y 的大作中提到】
: yes, indeed.
: does it has to be a folded domain? can it be some flexible linkers?
|
|
|
s******y
发帖数: 28562 | 11 Mmmmm, 谢谢，让我查查去。
【在 s******9 的大作中提到】
: E. coli ToA domain II, a very long alpha-helical domain and you can excise
: any length of fragments from it for your purpose. It serves as a spacer
: region for ribosome display.
|
b******y
发帖数: 627 | 12 If affecting the folding of flanking regions is your only concern, just use
a flexible linker.
On the contrary, I would worry about folding if you literally link two
compact domains without any linker. For instance, domain A ends with a helix
and domain B starts with a beta-strand. If you create an A-B polypeptide,
it is conceivable the last helix of A induces the first beta-strand into a
helix and therefore destabilizes the core of domain B.
【在 s******y 的大作中提到】
: 我担心的是假如linker 是flexible 的话会干扰到两端的蛋白折叠。
: 我的想法是如果linker 本身有很好的折叠的话应该会自己折起来就不会折到其他地方
: 去了。
|
n********k
发帖数: 2818 | 13 Yeah, Sunny, although I have no much knowledge about this shit:)), I can
back him up if he says so that positively.... as I know my soccer buddy well
. I am actually very jealous about him:))) our other soccer friend (HHMI)
said several time: bear is a true scientist and the best from our team that
have a number of faculty and several quasi-faculty in the pipeline, I have
one of the weakest if not the weakest records on the team members who
desire to stay academic...It is one of the great experience here to have
such a group of friends...
use
helix
【在 b******y 的大作中提到】
: If affecting the folding of flanking regions is your only concern, just use
: a flexible linker.
: On the contrary, I would worry about folding if you literally link two
: compact domains without any linker. For instance, domain A ends with a helix
: and domain B starts with a beta-strand. If you create an A-B polypeptide,
: it is conceivable the last helix of A induces the first beta-strand into a
: helix and therefore destabilizes the core of domain B.
|
b******y
发帖数: 627 | |
l**********1
发帖数: 5204 | 15 for what purpose? your final target
expression in Kock in Mice line?
【在 s******y 的大作中提到】
: 我们现在需要设计一个linker system, 需要在两个蛋白之间放进一串小
: 蛋白来调整那两个蛋白的距离。要求长度可调（就是可以靠放进蛋白的数量来
: 调节linker 的长度）。我知道别人做类似的事情的时候会放进titin I27 domain,
: 但是现在的问题是我们要求这个可以调节的单位长度必须在 50 Aa以内，而
: titin I27 domain正好比这个大一倍。
: 我不知道有什么蛋白的domain 是符合这个要求的？请大家推荐一下?就是必须能够
: 在５０Aa之内自发折叠成型（最好是球形），而且不要有太明显的功能区域（比方
: 说有个什么什么binding domain 就大大的不好）的东西。必须在５０个氨基酸内，
: 但也不要太短，6xHis 这种就太短了。２０～５０Aa最好。
: 谢谢！
|
s******y
发帖数: 28562 | 16 我的想法是用一连串的小蛋白做spacer, 但是小蛋白之间会放个大概6~8 Aa的flexible
linker, 总共会从1个单位到5个单位的样子（也就是最长可能达到300 Aa)
我担心假如放进一整串全部是flexible 的linker的话，如果太长的话（比方说一个
300Aa flexible linker） 就不知道会变什么样子。
use
helix
【在 b******y 的大作中提到】
: If affecting the folding of flanking regions is your only concern, just use
: a flexible linker.
: On the contrary, I would worry about folding if you literally link two
: compact domains without any linker. For instance, domain A ends with a helix
: and domain B starts with a beta-strand. If you create an A-B polypeptide,
: it is conceivable the last helix of A induces the first beta-strand into a
: helix and therefore destabilizes the core of domain B.
|
s******y
发帖数: 28562 | 17 表达在mammalian cell 里
【在 l**********1 的大作中提到】
: for what purpose? your final target
: expression in Kock in Mice line?
|
l**********1
发帖数: 5204 | 18 So should conside linked proteins pair its flolding or scafolding even
posstible proteasome by stuff E3 or other ubiquitn ligase etc problems
litte bear his opinion that is VIP. ----matrix one cent
【在 s******y 的大作中提到】
: 表达在mammalian cell 里
|
n********k
发帖数: 2818 | 19 你要做什么呀，这么复杂？我很好奇的说。
flexible
【在 s******y 的大作中提到】
: 我的想法是用一连串的小蛋白做spacer, 但是小蛋白之间会放个大概6~8 Aa的flexible
: linker, 总共会从1个单位到5个单位的样子（也就是最长可能达到300 Aa)
: 我担心假如放进一整串全部是flexible 的linker的话，如果太长的话（比方说一个
: 300Aa flexible linker） 就不知道会变什么样子。
:
: use
: helix
|
l**********1
发帖数: 5204 | 20 sunny day
是专利的话 有基金可以负担没?
pls refer:
Spacer: As used herein, the term "spacer" (also referred to as "linker")
refers to a peptide sequence between two protein moieties in a fusion
protein. A spacer is generally designed to be flexible or to interpose a
structure, such as an alpha-helix, between the two protein moieties. A
spacer can be relatively short, such as the sequence Gly-Ala-Pro (SEQ ID NO:
4) or Gly-Gly-Gly-Gly-Gly-Pro (SEQ ID NO: 5), or can be longer, such as,
for example, 10-25 amino acids in length.
Read more:
Patent application title: LYSOSOMAL TARGETING PEPTIDES AND USES THEREOF
Inventors: Jonathan H. Lebowitz (Whitefish Bay, WI, US) John Maga (
Whitefish Bay, WI, US)
Assignees: Zystor Therapeutics, Inc.
IPC8 Class: AA61K3843FI
USPC Class: 424 943
Class name: Drug, bio-affecting and body treating compositions enzyme or
coenzyme containing stabilized enzymes or enzymes complexed with nonenzyme (
e.g., liposomes, etc.)
Publication date: 2011-09-15
link:
//www.faqs.org/patents/app/20110223147#b
【在 l**********1 的大作中提到】
: So should conside linked proteins pair its flolding or scafolding even
: posstible proteasome by stuff E3 or other ubiquitn ligase etc problems
: litte bear his opinion that is VIP. ----matrix one cent
|
l**********1
发帖数: 5204 | 21 N 院士 站内PM你了
---just for credit protection (possible)
【在 n********k 的大作中提到】
: 你要做什么呀，这么复杂？我很好奇的说。
:
: flexible
|
