Dear Shaheera Fatima

you can certanilly used same linkers between more sequences. Personally i suggest to you to differentiate it in terms of DNA codons so in case later on you would like to modify your clone by PCR (for example performing an insertion or deletion) in a region close to the linker you have less problem of primers specificity, that may happen is the sequence of the linker is included in the primer and the linkers are codified from the same bases.

Which is the best linker (eg leght, presence or abcence) is difficult and may change from protein to protein on the basis of protein rigidity and steric hydrance (for example if you connenct to big and rigid proteins longer linkers (eg GSGGGG insteat GS) may be preferable since the proteins are more free to move, on the other hand a long linker could be more susceptible to protease digestion.

The same evaluation can be true for the His tag, mantain a linker beetween the tag and protein can favour TAG correct exposition and than protein purification but in this way you had some uinwanted AA.

There is no a best choice. You have to try the one that you like more and be reasy to change your strategy if this will not work.

Personally fro protein with N-term his tag i like also the idea to insert the TEV cleavege site as a linker after the His tag which will allow to you to remove the His tag and the linker, peforming TEV digestion and a 2nd step of subctractive IMAC purification in case that linker and his tag may affect your downstream applications.

in case you are interested, you can find some more information about subttractive IMAC in the follwoing link

https://www.blogger.com/video.g?token=AD6v5dxhCeJUPx5UODBu4Y9hhJZix_l5JZcPb03ZUe7G3qL8GhoPUWZMm6z2wEJJED9OY8gn_ttNXrGNCdVurZ6zkB_oTSCrmRc90-ux7AQAOhaddCkXKREYmgNw87RSh58zQ4uVQVtz

good luck

Manuele

I want to display the whole sequence on the cell's surface. I do not want to cleavage it for purification. In this case which linker is suitable for joining the sequences?