pRSET expression system troubles
legatek at mcmail.cis.mcmaster.ca
Mon Aug 19 15:50:17 EST 2002
On 19 Aug 2002, Wolfgang Schechinger wrote:
> I have been using pRSET B. Worked fine with balb/3t3 and 293 HEK cells
> (transfection with PEI and Lipofectamine).
I don't know how this can be since pRSET vectors are prokaryotic His-tag
I have used pRSET B in the BL21 salt inducible system with
variable success. One of my proteins expresses and purifies extremely
well, a point mutant expresses much lower and doesn't purify as well, and
two other point mutants express very well and don't purify at all.
If your protein doesn't express it may be due to: 1. Your protein
hasn't been cloned in frame, 2. Your protein is toxic/unstable, 3. Your
cells don't have T7 polymerase. Sequence the clone, make sure you're using
an appropriate strain for overexpression, and fiddle with the growth
conditions. After comparison of BL21(DE3) and BL21SI I settled on the salt
inducible system, at 30 degreesC. Expression was poor at 37C.
I hope this helps.
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legatek at mcmaster.ca Kyle Legate legatek at hotmail.com
Tower of Tongues:Thursday PM:10:30-11:30 EDT:http://cfmu.mcmaster.ca
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