pRSET expression system troubles

Wolfgang Schechinger marsupilamiwolfgang at web.de
Mon Aug 19 17:21:27 EST 2002


Oops, 

Kyle, you're right. I messed pRSET up with pSecTag2. Anyway, your
suggestions are right. 

Wo

At 16:50 19.08.02 -0400, Kyle Legate wrote:
>On 19 Aug 2002, Wolfgang Schechinger wrote:
>
>> Joe,
>> 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
>vectors.
>	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.
>
>... . . .  .  .  .    .    .    .     .     .     .      .      .      .
>legatek at mcmaster.ca		Kyle Legate            legatek at hotmail.com
>
>   Tower of Tongues:Thursday PM:10:30-11:30 EDT:http://cfmu.mcmaster.ca
>    moon musick:ritual:IDM:experimental(electronica):minimalism:glitch
>.      .      .     .     .     .    .    .    .   .   .   .  .  . . ...
>
>
>

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