GlyGlyGlySer can be used to introduce a flexible linker.

Hi,

In your described construct, N terminus-MBP-His-ThrombinSite-Protein I see an immediate issue. The His tag is buried in the middle after MBP. The His tag is less likely to be accessible to the nickel column at that location in your protein. Remove MBP or put the His tag on the N-terminus followed by MBP.

I agree with Jeffrey, in that I would put the His-tag on the N-terminus of your construct, but others have been successful with your arrangement, and the pET 32a vector is setup the same way as your construct, you may want to take a look at the following RG discussion on the same topic:

https://www.researchgate.net/post/Has_anyone_ever_placed_a_His_tag_between_two_fusion_proteins_and_got_great_purification

I also agree with Jeffrey. Beware; if you want to cleave the tag, thrombin cleavage works out terrible. Factor Xa is a better option if you don’t have internal sites, TEV or PreScission even better.

Thanks everyone. After your suggestions, I switched to MBP-TEV-protein construct. Are there any suggestions for a linker between the MBP and the TEV site and between the TEV site and the protein? Thanks again.

I do not include any linker sequences between tag and protein of interest. It works well for almost all common downstream experiments like affinity chromatography, co-ip, western, etc.

There is actually no need for it, am only concern about the position of your His-Tag and its eventual purification. why not move it to the start the N-terminal or end the C-terminal? then Carola is right with the thrombin cleavage. please consider TEV instead