I am part of an undergraduate research group and we are trying to produce a protease in an E.coli gene circuit. We are not sure where to place the His tag in our circuit. The sequence is ...OmpA(gene)->linker->his tag->protease->Terminator->res site Should the His tag be in this position, before the linker, or after the protease? OmpA is a outer membrane protein that will allow a protein to be taken to, and linked to, the outer membrane of the E.coli. Our fear is that the protease will not fold correctly and will be nonfunctional.

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    $\begingroup$ When you say gene, have you made sure that the stop codon is removed? $\endgroup$ – WYSIWYG Jun 24 '15 at 15:55
  • $\begingroup$ Usually the his-tag should be in the terminal. Why don't you express the protease as a separate cistron? $\endgroup$ – WYSIWYG Jun 24 '15 at 15:57
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    $\begingroup$ There are advantages and disadvantages to put His-tag both N and C termini. When you put a tag at a C terminus, you can purify the full length easily. If you are lucky, you could get full length products even if you put a tag at the N terminus. An additional sequence at a C-terminus may disturb functions of some proteins, but in most of cases, it would not be predictable. $\endgroup$ – 243 Jun 24 '15 at 16:11
  • $\begingroup$ We have removed the stop codon of the gene. We wanted to have the His tag attached to the protease in some way to use as proof of concept that they protease is being expressed on the outer membrane of the E.coli. $\endgroup$ – Kelly Eckartt Jun 24 '15 at 16:17
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    $\begingroup$ Then what, I think, you should do is put his-tag after the protease. Is the C-terminal of Omp-A on the outside? $\endgroup$ – WYSIWYG Jun 24 '15 at 16:21

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