w******e
发帖数: 1187 | 1 让我对droplet/IVC的饭度大减呵呵。跟phage比也有definitive的优势——
毕竟是HT screening而不是selection。我小见识浅薄一惊一乍大家别见怪呵呵。
ms就wittrup一枝独秀？国内有phage/yeast display的牛人吗？
内行指点一下？ |
s******9
发帖数: 283 | 2 问几个问题。好奇yeast display能稳定表达多大的protein？Phage display好像只能
表达不大的peptide。Microfludics做IVC有多成熟？是不是都只有fluorescence
readouts？
【在 w******e 的大作中提到】
: 让我对droplet/IVC的饭度大减呵呵。跟phage比也有definitive的优势——
: 毕竟是HT screening而不是selection。我小见识浅薄一惊一乍大家别见怪呵呵。
: ms就wittrup一枝独秀？国内有phage/yeast display的牛人吗？
: 内行指点一下？
|
s******9
发帖数: 283 | 3 还有，象ribosome/mRNA display这些single-molecule based display和yeast/phage
display在传统的screening环节相比是劣势还是优势？
【在 s******9 的大作中提到】
: 问几个问题。好奇yeast display能稳定表达多大的protein？Phage display好像只能
: 表达不大的peptide。Microfludics做IVC有多成熟？是不是都只有fluorescence
: readouts？
|
l******a
发帖数: 3339 | 4 你是说yeast surface display？貌似以前读过一篇review，比起phage之类有很大的
technical challenge，不是人人都能做的。
【在 w******e 的大作中提到】
: 让我对droplet/IVC的饭度大减呵呵。跟phage比也有definitive的优势——
: 毕竟是HT screening而不是selection。我小见识浅薄一惊一乍大家别见怪呵呵。
: ms就wittrup一枝独秀？国内有phage/yeast display的牛人吗？
: 内行指点一下？
|
l*********1
发帖数: 351 | 5 要做大蛋白就用mrna或者ribosome display.不需要优化phage或者yeast 表面的蛋白表
达.
缺点就是贵,很贵,二screening稍微麻烦点,时间稍长.
【在 s******9 的大作中提到】
: 问几个问题。好奇yeast display能稳定表达多大的protein？Phage display好像只能
: 表达不大的peptide。Microfludics做IVC有多成熟？是不是都只有fluorescence
: readouts？
|
l*********1
发帖数: 351 | 6 国内有人开始用ribosome display了.华西医学院不是有个教授做抗体工程还不错的.
赚钱就靠这个方向了.
【在 w******e 的大作中提到】
: 让我对droplet/IVC的饭度大减呵呵。跟phage比也有definitive的优势——
: 毕竟是HT screening而不是selection。我小见识浅薄一惊一乍大家别见怪呵呵。
: ms就wittrup一枝独秀？国内有phage/yeast display的牛人吗？
: 内行指点一下？
|
s******9
发帖数: 283 | 7 mRNA display很贵，ribosome display还好吧。
【在 l*********1 的大作中提到】
: 要做大蛋白就用mrna或者ribosome display.不需要优化phage或者yeast 表面的蛋白表
: 达.
: 缺点就是贵,很贵,二screening稍微麻烦点,时间稍长.
|
s******9
发帖数: 283 | 8 好像抗体还是phage display用得多， 所以我好奇ribsome display在screening相比上
肯定会有弱点。
【在 l*********1 的大作中提到】
: 国内有人开始用ribosome display了.华西医学院不是有个教授做抗体工程还不错的.
: 赚钱就靠这个方向了.
|
x*****e
发帖数: 309 | 9 技术上还行吧，我以前做过，酵母专染效率比较低，10e3左右，建库工作量大不少。我
记的MIT有个人做yeast display
【在 l******a 的大作中提到】
: 你是说yeast surface display？貌似以前读过一篇review，比起phage之类有很大的
: technical challenge，不是人人都能做的。
|
H****s
发帖数: 301 | 10 It is not as powerful as what you thought. Every display technology has its
cons and pros, so is yeast surface display. The good part about yeast
surface display is that, combined with FACS, throughput can be significantly
increased. However, good display does not necessarily mean that the
displayed protein is functional.
【在 w******e 的大作中提到】
: 让我对droplet/IVC的饭度大减呵呵。跟phage比也有definitive的优势——
: 毕竟是HT screening而不是selection。我小见识浅薄一惊一乍大家别见怪呵呵。
: ms就wittrup一枝独秀？国内有phage/yeast display的牛人吗？
: 内行指点一下？
|
|
|
H****s
发帖数: 301 | 11 Now for yeast transformation, you can reach an efficiency up to 10e10.
【在 x*****e 的大作中提到】
: 技术上还行吧，我以前做过，酵母专染效率比较低，10e3左右，建库工作量大不少。我
: 记的MIT有个人做yeast display
|
w******e
发帖数: 1187 | 12 I remember you are the expert on this. got a question for you: is yeast
display officially a better method for affinity maturation now?
some work from Wittrup group is amazing...
its
significantly
being FACSable is a big deal to me-- quantitative, monitoring individual
clone
that's a problem for every display I guess, or do you mean it's a much
bigger concern for yeast?
【在 H****s 的大作中提到】
: It is not as powerful as what you thought. Every display technology has its
: cons and pros, so is yeast surface display. The good part about yeast
: surface display is that, combined with FACS, throughput can be significantly
: increased. However, good display does not necessarily mean that the
: displayed protein is functional.
|
w******e
发帖数: 1187 | 13
not true on PIII. scFv is the tour de force for phage hehe
there are many fancy types, but fluorescence is so nice why change?
btw many other displays also do fluorescence.
【在 s******9 的大作中提到】
: 问几个问题。好奇yeast display能稳定表达多大的protein？Phage display好像只能
: 表达不大的peptide。Microfludics做IVC有多成熟？是不是都只有fluorescence
: readouts？
|
w******e
发帖数: 1187 | 14
phage
very tech sensitive
【在 s******9 的大作中提到】
: 还有，象ribosome/mRNA display这些single-molecule based display和yeast/phage
: display在传统的screening环节相比是劣势还是优势？
|
w******e
发帖数: 1187 | 15 how come? doesn't ribosome display need cell-free expression system too?
that's the most expensive part bah
【在 s******9 的大作中提到】
: mRNA display很贵，ribosome display还好吧。
|
l******a
发帖数: 3339 | 16 cell extract 就可以吧，如果不加非自然氨基酸的话。concept上听着还挺simple的，
不知道具
体如何。
too?
【在 w******e 的大作中提到】
: how come? doesn't ribosome display need cell-free expression system too?
: that's the most expensive part bah
|
H****s
发帖数: 301 | 17 Not an expert on this topic, but I am willing to share my 2 cents.
For antibody affinity maturation, yeast display is better than other
technologies. FACS sorting, combined with yeast display, increases the
throughput significantly. You can quantify binding affinity and kinetics
with flow cytometry. At the same time, you do not need to work with a
library larger than 10e10, which is still a pain in the field. Dr. Wittrup
established a company in New Hampshire. The name is Adimab. You can check
their technologies.
However, there are still a lot of problems. (1). Poor display level. (2).
Poor correlation between display level and activity. (3). Intellectual
property issues.
【在 w******e 的大作中提到】
: I remember you are the expert on this. got a question for you: is yeast
: display officially a better method for affinity maturation now?
: some work from Wittrup group is amazing...
:
: its
: significantly
: being FACSable is a big deal to me-- quantitative, monitoring individual
: clone
: that's a problem for every display I guess, or do you mean it's a much
: bigger concern for yeast?
|
w******e
发帖数: 1187 | 18 Thanks for the input.
en the variability on display level is indeed daunting.
BTW why didn't bacteria display take off? bacteria can do FACS too,
right? plus I assume it's easier to maintain.
Also, IP issues seem to really hurt protein engineering in general,
according to Bradbury's recent review. I assume phage display is
even worse? considering there are mutliple big shots hehe
【在 H****s 的大作中提到】
: Not an expert on this topic, but I am willing to share my 2 cents.
: For antibody affinity maturation, yeast display is better than other
: technologies. FACS sorting, combined with yeast display, increases the
: throughput significantly. You can quantify binding affinity and kinetics
: with flow cytometry. At the same time, you do not need to work with a
: library larger than 10e10, which is still a pain in the field. Dr. Wittrup
: established a company in New Hampshire. The name is Adimab. You can check
: their technologies.
: However, there are still a lot of problems. (1). Poor display level. (2).
: Poor correlation between display level and activity. (3). Intellectual
|
H****s
发帖数: 301 | 19 好孩子，你还是看了我推荐的那些人写的综述的。之所以bacterial display不行，是
因为能够functionally display在细菌表面的蛋白真是太少了。如果你用细菌表达过真
核蛋白，你应该会明白其中的难处。正确的蛋白折叠、可溶性、毒性对于bacterial
display来说是个很大的挑战。酵母不存在此等问题，但酵母的问题在于糖基化修饰。
保持联系吧，说不定以后我能帮你点小忙。
【在 w******e 的大作中提到】
: Thanks for the input.
: en the variability on display level is indeed daunting.
: BTW why didn't bacteria display take off? bacteria can do FACS too,
: right? plus I assume it's easier to maintain.
: Also, IP issues seem to really hurt protein engineering in general,
: according to Bradbury's recent review. I assume phage display is
: even worse? considering there are mutliple big shots hehe
|
w******e
发帖数: 1187 | 20 嘿嘿，偷师被发现了：）
【在 H****s 的大作中提到】
: 好孩子，你还是看了我推荐的那些人写的综述的。之所以bacterial display不行，是
: 因为能够functionally display在细菌表面的蛋白真是太少了。如果你用细菌表达过真
: 核蛋白，你应该会明白其中的难处。正确的蛋白折叠、可溶性、毒性对于bacterial
: display来说是个很大的挑战。酵母不存在此等问题，但酵母的问题在于糖基化修饰。
: 保持联系吧，说不定以后我能帮你点小忙。
|
T**********t
发帖数: 1604 | |
H****7
发帖数: 159 | 22 I just wonder where you got those information, is it Ok to share with us?
Thanks. A
【在 H****s 的大作中提到】
: 好孩子，你还是看了我推荐的那些人写的综述的。之所以bacterial display不行，是
: 因为能够functionally display在细菌表面的蛋白真是太少了。如果你用细菌表达过真
: 核蛋白，你应该会明白其中的难处。正确的蛋白折叠、可溶性、毒性对于bacterial
: display来说是个很大的挑战。酵母不存在此等问题，但酵母的问题在于糖基化修饰。
: 保持联系吧，说不定以后我能帮你点小忙。
|
