Hello everyone!
I was wondering how to go about expressing some full-length proteins that I will need active. I read somewhere that an 6xHis-MBP tag might be good. Some people in lab have GST or 6xHis and express in E.Coli DE3 pLys. I have no experience with this but a cleavable 6xHis-MBP recombinant protein sounds good. Uhm........ perhaps someone more experienced might have other suggestions I could look into?
All I know is I need the proteins active and I might have to to introduce mutations to compare activity at some point.
Thank you and have a nice day!
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#2
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Posted 21 December 2010 - 08:00 AM
I would use E. coli if at all possible. Insect cells are tougher to maintain.
#3
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Posted 21 December 2010 - 10:45 PM
Hola, in my opinion the expression system will be dependent of the complexity of protein and the postranslational modifications. In the kinases, the PIM family around 30Kd is well expressed in E.coli, soluble and with activity.In the case of the PI3K complex 110 and (85 or 30Kd) if you try in E coli, probably express it in inclusion bodies, and renaturalization probably will give low yield of active protein. One more fact,all the active commercial human proteins are expresed in insect cells with 6His-or GST tags. There are some pages in internet which predicts the solubility of your recombinant protein after introducing the sequence. I would know the % of probability of solubility and if it were high, I will try in bacteria, if no probably it would insoluble in any system but I would try a secretion system. Probably more forers will give you more ideas better than mines. Buena suerte y Feliz Navidad
