1 reply to this topic

[DNZC]

Hi all,

I tried to express a peripheral membrane protein in E coli system.  When I insert the gene into the pET22b vector (NdeI/BamHI site), there is no expression when I use BL21(DE3), BL21(DE3)pLysS, Rosetta(DE3), C41, Lemo21. I extracted the vector from E coli and got it sequenced. So the vector containing the correct gene sequence is well transformed.  However, when I insert the gene into pMAL-c5g, the MBP-fused protein is highly expressed. Is there any possible way to express the protein without the MBP fusion? Because the fusion protein, like MBP or GST, also binds the metal, which is not desired. Thanks you guys~~

[protolder]

DNZC, on 23 July 2011 - 05:50 PM, said:

Hi all,

I tried to express a peripheral membrane protein in E coli system.  When I insert the gene into the pET22b vector (NdeI/BamHI site), there is no expression when I use BL21(DE3), BL21(DE3)pLysS, Rosetta(DE3), C41, Lemo21. I extracted the vector from E coli and got it sequenced. So the vector containing the correct gene sequence is well transformed.  However, when I insert the gene into pMAL-c5g, the MBP-fused protein is highly expressed. Is there any possible way to express the protein without the MBP fusion? Because the fusion protein, like MBP or GST, also binds the metal, which is not desired. Thanks you guys~~

Hola for me the main differennce between both expression vectors is the lac repressor, lacI in in pET but lacIq (stronger)in pMal. So I´LL try to compensate this repression using a lacIq strain as any Origami, Rosetta-gami or NovaBlue or any strain that carries this lacIq repressor often in episome in F+ strains. Buena suerte