n****e
发帖数: 1677 | 1 I used to make different constructs for 1 protein, they are all soluble, but
behave different, some are monomer, inactive, some are dimer inactive, some
are dimer active can't crystallize, some are dimer active can
crystallize......
And the difference between these constructs are just around 10 residues.
所不
另一
而不
来就 | v**a
发帖数: 107 | 2
~~~~~~~~~~~
this difference is already big enough, for my protein, a Ser-->Ala will kill
it all.
I am not sure I know you situation well, but I would suggest you to focus on
one
construct at a time or plan your constructs with less different residues. I am
working on enzyme mechanism, so I am very allergic to point mutations, hehe.
10 residues
different at active site with no significant harm to activity, for me, that is
a miracle.
o
【在 n****e 的大作中提到】
: I used to make different constructs for 1 protein, they are all soluble, but
: behave different, some are monomer, inactive, some are dimer inactive, some
: are dimer active can't crystallize, some are dimer active can
: crystallize......
: And the difference between these constructs are just around 10 residues.
:
: 所不
: 另一
: 而不
: 来就
| n****e
发帖数: 1677 | 3 可能我说的不太清楚，我解释一下，在表达一个蛋白质前，首先要做的是blast,
找到保守序列，然后设计constructs，比如我做的蛋白，保守序列是1500-2183，
那我这样设计construct,
N-terminal:1400,1409,1419,1429,1439,1449
C-terminal:2233,2223,2213,2203
这些加一起可以做24个constructs,我表达的结果1429-2233表达，很稳定，dimer,active
,crystallized,然后crystal structure表明在N-terminal和
C-terminal各有几十个残基disorder,然后我就在crystal structure基础上
再设计一些constructs,把disorder的残基cut掉，结果我又表达了几个
constructs,包括1476-2233,1476-2189,1495-2233,1495-2189等等。然后结果
就比较有趣了。
上面四个都表达可溶稳定，不同的是，1476-2233,1495-2233是dimer,active,
1476-21 | t****u
发帖数: 10218 | 4 I wasn't talking about the crystal structure either.:)
I thought in the pulldown exp. you had a GST fusion to pull-down another
version of your protein, is that right? I am concerning about that your
GST-fusions are so geographically close to each other there will be very
little chance to form a GST-protein/protein dimer. Most of them will be
GST-protein/GST-protein dimer, that's why "E coli. 表达纯化的yeast蛋白不能形成
二聚体 （GST pulldown)".
But I'm just speculating, didn't know your exp. detail.
maybe
的修饰 |
|
