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Vector with T5-promotor in Bl21/DE3-system


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#1 Linnea

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Posted 01 March 2005 - 07:05 AM

I'm expressing a His-tagged protein of 4.5 kDa in E.Coli BL21/DE3-cells. I'm using the pQE30 FXa vector from Qiagen, in which the T5-promotor regulates expression (addition of IPTG induces expression).

I've got indications of the plasmid being leaky, that is no IPTG needs to be added to get protein expression.

Has anyone else experience of a T5-promotor regulated vector behaving oddly in BL21/DE3 cells or any other cells?

I would like to know as it would explain a great deal of my results. Otherwise I have extensive troubleshooting to do.

I greatly appreciate any help i can get!

#2 Linnea

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Posted 21 March 2005 - 06:00 AM

I found out why the expression of a gene inserted in the pQE-30 FXa vector wasn't efficiently expressed in BL21/DE3 cells. The polymerase (T5) repressor isn't included in the vector. You need a E.Coli strain that carries it, for example M15[prep4].




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