3 replies to this topic

[Sharky]

Hey guys,
            So I usually get helpful advice on this forum, so here goes!

1) I expressed my gene with a his tag in BL21(DE3) at 30 degrees with 1mM IPTG. = Saw a good expression band on the SDS-page at 3 hours post induction with majority being soluble.
2) I expressed the same gene but without his tag in BL21(DE3) at 30 degrees with 1mM IPTG = Saw no exression whatsoever in both soluble and insoluble fractions.

What is going on? I am aware of his tag causing insolubility problems, but is it possible that a his tag is beneficial for this protein from an expression/solubility sense?

Attachment 1 : Gene 1 with tag. showing expression in the middle lanes(mid gel).
Attachment 2  : Gene 1 without tag showing no expression

Attached Thumbnails

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[sdar]

i dont know the solubility effect but i know that his tag does stabilizes the protein so it is better to use it

[pDNA]

the intensity of all bands seem to in increased in your induced samples ...this is somehow strange?

Maybe you can indicate what band on the gel is your "overexpressed" gene?

Regards,
p

[HomeBrew]

How did you clone your inserts?  If by PCR, perhaps the clone without the his-tag has an incorporation error in it that's causing it not to be expressed.  Also, did you check orientation of the non-his-tagged clone's insert?