s*******m
发帖数: 24 | 1 需要大量纯化一个小蛋白，大概9KD左右。在大肠杆菌里面表达量一直不高。以前看过
一篇文章，说需要在蛋白N端第2个或者第3个氨基酸做一个突变，可以提高表达量。有
谁知道是什么突变不？或者有谁知道怎么在细菌里面大量表达小蛋白的办法不？多谢了
！ | h**********r
发帖数: 671 | 2 难道是ACP？瞎猜的～～～～没有做过小蛋白的表达。不过看paper提到下面一些策略，希望有点帮助：
1. Introduce an Ala codon (GCU) the second position of the sequence
2. Increase the AT content of the 5’-end, particularly codons 4 and 5, by
silent mutagenesis. This reduces the potential for mRNA secondary structure,
which may inhibit ribosome binding/translation. The studies identified a
distinct nucleotide preference for this region (A > T > G > C).
3. If possible, the preferred stop sequence (TAAT).
改变第二位氨基酸为Ala见得比较多。做过统计，在e. coli里偏好ALa. | s*******m
发帖数: 24 | 3 Is the Ala trick specific to small protein? I remembered that there is a
mutation specific to small protein expression in E coli.Just can't find the
paper anymore.....
，希望有点帮助：
structure,
【在 h**********r 的大作中提到】
: 难道是ACP？瞎猜的～～～～没有做过小蛋白的表达。不过看paper提到下面一些策略，希望有点帮助：
: 1. Introduce an Ala codon (GCU) the second position of the sequence
: 2. Increase the AT content of the 5’-end, particularly codons 4 and 5, by
: silent mutagenesis. This reduces the potential for mRNA secondary structure,
: which may inhibit ribosome binding/translation. The studies identified a
: distinct nucleotide preference for this region (A > T > G > C).
: 3. If possible, the preferred stop sequence (TAAT).
: 改变第二位氨基酸为Ala见得比较多。做过统计，在e. coli里偏好ALa.
| s*******m
发帖数: 24 | | h**********r
发帖数: 671 | 5 母鸡小蛋白啊。没准儿你要表达的小蛋白有特殊的生理功能，就是难高表达。换不同的
宿主试试 | f*c
发帖数: 2726 | 6 probablly n-end rule
(no arginine, lysine, leucine, phenylalanine, tyrosine, and tryptophan);
Met, Gly, Ala, Ser, Thr, Val, Pro may be better
【在 s*******m 的大作中提到】
: 需要大量纯化一个小蛋白，大概9KD左右。在大肠杆菌里面表达量一直不高。以前看过
: 一篇文章，说需要在蛋白N端第2个或者第3个氨基酸做一个突变，可以提高表达量。有
: 谁知道是什么突变不？或者有谁知道怎么在细菌里面大量表达小蛋白的办法不？多谢了
: ！
| b******y
发帖数: 627 | | b******y
发帖数: 627 | 8 If possible, use a N-terminal tag, e.g. MBP. I bet you will have a lot of
protein expressed. | s*******m
发帖数: 24 | 9 以前有一篇文章，专门讲小蛋白的。据说一般情况下，小蛋白在细菌里面表达量都比较
低。好像是因为容易被降解。但是要是在第2或者第3个氨基酸的位置放一个特定的氨基
酸，就可以保护蛋白不被降解。不过我怎么也找不到那个文章了。。。。
【在 h**********r 的大作中提到】
: 母鸡小蛋白啊。没准儿你要表达的小蛋白有特殊的生理功能，就是难高表达。换不同的
: 宿主试试
| b******y
发帖数: 627 | 10 I have expressed a number of small proteins or small protein domains or even
peptides in E. coli. There were no problem for expression or purification.
Most of them have His-tag, of course.
For the ones that His-tag does not work well, I will use a MBP tag and 90%
of the cases will be solved as long as the proteins have some expression in
their naked forms. | g********4
发帖数: 4959 | 11 How do you check your protein? By comossie stain? I know Ubiquitin is
somehow resistant to check by this method, and actually, it is expressed
quite well in e coli. |
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