Thread: how to introduce a his-tag in a protein complex

Hi,

I want to clone the subunit E with a histidine tag from complex I of E. coli (thought to amplify the gene with primers that introduce the tag). Then replace the wild-type gene to this subunit with the tag in the genome to purify the entire complex with an histidine-tag.

I thought to amplify the flanking regions and clone each one to a vector. Amplify the gene with primers that introduce the tag. Then introduce this gene with his-tag between flanking regions and use the pKO3 system to introduce the construct in E. coli genome.

But I don´t know if this the best strategy. Im also having problems with primer design (restrition enzymes, transcription start, etc) and how all of this can affect my complex I... and mostly how to put the gene in the proper orientation.

If you can give some ideias how to overcome this problems I would be very grateful ( Im relatively new in cloning )

thanks,

Andreia