w*********e
发帖数: 98 | 1 想在大肠杆菌中表达一个N末端GST融合的真核蛋白。但是表达出的都是C端缺失的蛋白
。因为 用GST antibody 可以检测到非全长的蛋白条带.请问如何改善条件才能获得全
长蛋白？多谢！ |
n***w
发帖数: 2405 | 2 just to be curious, how do you know the c-terminus is missing? Using
antibody targeting c-terminus?
Have you checked the sequence to see it's in the right open reading frame?
Is that the bacteria have some protease to chop the protein off?
Alternatively, you can turn to baculovirus expression. But you need to
change the whole system in that case.
I am doing the same thing and being frustrated.... |
w*********e
发帖数: 98 | 3 It is a N-terminal GST fused protein. I did western using GST antibody and
detected a smaller size band.
【在 n***w 的大作中提到】
: just to be curious, how do you know the c-terminus is missing? Using
: antibody targeting c-terminus?
: Have you checked the sequence to see it's in the right open reading frame?
: Is that the bacteria have some protease to chop the protein off?
: Alternatively, you can turn to baculovirus expression. But you need to
: change the whole system in that case.
: I am doing the same thing and being frustrated....
|
C*******e
发帖数: 4348 | 4 GST表达的蛋白好像挺容易产生GST only的，
25 kDa左右
你有试过别的tag么？
比如N-his
或者C-his
and
【在 w*********e 的大作中提到】
: It is a N-terminal GST fused protein. I did western using GST antibody and
: detected a smaller size band.
|
w*********e
发帖数: 98 | 5 The band is bigger than GST. It is around half size of the full lengh
protein. I am thinking that probably the sequence of the protein is not
easy for bacteria to translate. I am wondering if there are some conditions
could overcome this problem.
【在 C*******e 的大作中提到】
: GST表达的蛋白好像挺容易产生GST only的，
: 25 kDa左右
: 你有试过别的tag么？
: 比如N-his
: 或者C-his
:
: and
|
n***w
发帖数: 2405 | 6 If you use pGEX2T vector, then the GST is about 29kDa. If your observed
differences were within the 3-4 kDa range, then it may be fine.
Also, you can use some online tools to see if your proteins have rare amino
acids or not. Codon bias may exist. |
n***w
发帖数: 2405 | 7 If codon bias does exist, as someone here suggested, you can use Rosetta-gami series competent cells or
other strains that are capable to translate rare amino acids.
Besides GST-tag antibody, try some antibodies that target your protein. Then you may have a better picture
what is going on here.
I met almost the same problems as you. First I found I didn't use the right strain. Then I realized my sequence
contains many rare amino acids. |
w*********e
发帖数: 98 | 8 Do you have any link for checking rare amino acids? Thanks
amino
【在 n***w 的大作中提到】
: If you use pGEX2T vector, then the GST is about 29kDa. If your observed
: differences were within the 3-4 kDa range, then it may be fine.
: Also, you can use some online tools to see if your proteins have rare amino
: acids or not. Codon bias may exist.
|
n***w
发帖数: 2405 | 9 http://nihserver.mbi.ucla.edu/RACC/
【在 w*********e 的大作中提到】
: Do you have any link for checking rare amino acids? Thanks
:
: amino
|
w*********e
发帖数: 98 | 10 Thank you so much! I am going to check now.
【在 n***w 的大作中提到】
: http://nihserver.mbi.ucla.edu/RACC/
|
C*******e
发帖数: 4348 | 11 如果是codon bias
可以
1.用某些特殊的BL21(DE3)菌株，具体不记得哪些选择了，自己去搜一下
2.实在不行可以codon optimize，换成ecoli喜欢的codon
conditions
【在 w*********e 的大作中提到】
: The band is bigger than GST. It is around half size of the full lengh
: protein. I am thinking that probably the sequence of the protein is not
: easy for bacteria to translate. I am wondering if there are some conditions
: could overcome this problem.
|
T********e
发帖数: 223 | |
