I noticed that you could get some soluble protein in supernatant(~10-15%). Therefore, it becomes important to think about what is the purpose to purify this protein.
If you want to do crystallization or NMR ，maybe it is worth to try to improve the soluble expression. But I strongly suggest to obtain enough protein simply by increasing the culture volume.
If you want to do some biochemistry experiments, it will definitely waste your time to search methods improving the soluble expression.作者: star#room 时间: 2013-4-24 16:25
I noticed that you could get some soluble protein in supernatant(~10-15%). Therefore, it becomes important to think about what is the purpose to purify this protein.
If you want to do crystallizati ...
This protein is an enzyme that I want to reserch the catalytic activity. On the other hand, degeneration and renaturation of inclusion body is very difficult for my enzyme(I have tried).So I want to get enough soluble protein for purification. Whicht the common vector can decrease the amount of inclusion body and increase the soluble protein? What about pMAL vector? Thank you !作者: hot_hot_hot 时间: 2013-4-24 16:25
This protein is an enzyme that I want to reserch the catalytic activity. On the other hand, degeneration and renaturation of inclusion body is very difficult for my enzyme(I have tried).So I want to ...
According to my limited experience, the problems in expressing heterogenes, such as expressing or not, soluble or not, are mostly depending on the intrinsic characterization of this protein. Occasionally, the expression level could be improved by adjusting in vivo (for EX. vector, host, code bias, codon optimization, truncation expression) or in vitro (medium, temp., air, agitation, inducer) conditions. It is very difficult to say which vector is the best. The only thing you can do is to try one by one. Of course, pMAL should be tried. According to the manufacturer, MBP will certainly improve the expression, but they have never guaranteed.作者: star#room 时间: 2013-4-24 16:26
Thank you for your advice. I will try another host which can express thioredoxin reductase and glutathione reductase. If the result are not good, I think I will change the vector or express my gene in Saccharomy cescerevisiae.作者: nut6694 时间: 2013-4-24 16:27
QUOTE:
原帖由 star#room 于 2013-4-24 16:26 发表 bbcodeurl('http://bbs.antpedia.com/images/common/back.gif', '%s')
Thank you for your advice. I will try another host which can express thioredoxin reductase and glutathione reductase. If the result are not good, I think I will change the vector or express my gene in ...
