I have a plasmid that I inherited from a previous lab mate. It is his6-thrombin-POI-KLAAALE-his6. I couldn't figure out why KLAAALE is needed as the linker peptide on the C-terminus but I saw multiple papers use this technology. Could anyone provide some insight in this?
Hi Peng Ge,
Do you know into which vector backbone this insert was cloned into, using which cloning method and restriction sites? The last two residues (LE) may stem from an XhoI restriction site used for cloning. The other residues may likewise stem from the multiple cloning site in the vector backbone.
Hi,
Bernhard Lechtenberg is I believe correct in that the amino acid sequence is actually a remnant of the multiple restriction site. For instance, if you look at the original sequence of pET28a, KLAAALE precedes the C-terminal his-tag and encompasses HindIII, NotI, EagI, XhoI and other restriction sites.
Hi there,
As Ana mentioned this peptide is the result of the translation of the remaining of the MCS. The cloning has been obviously done using HindIII site as the downstream cloning site (translation of HindIII site AAGCTT gives KL).
Bernhard Lechtenberg Thanks!!! It makes sense. This sequence should be resulted from the vector. It has been so many years since the plasmid was constructed. I am actually not quite sure which vector it was. All I know is kanamycin-resistant. Also, I'm wondering at which AA point BL21 will stop producing with my POI? I mean, will my POI be produced with the KLAAALE-His6 sequence? Is it possible to stop it right after the last AA of my protien?
If there is no stop codon there is no real chance to stop translation... You should go for plasmid sequencing to get sure about what is actually present btween promoter and terminator.
- Badges
- Science topic