Plasmid #10 allows for the purification of membrane proteins. It includes an N-terminal His8 tag, a C-terminal CL7 tag, and an engineered signal peptide from the Bacillus halodurans YidC gene. Membrane proteins are inserted at the BamHI/SpeI restriction sites, where there is an existing 1711 base pair restriction fragment.
Transcription is induced with IPTG and driven by the T7 RNA polymerase. The plasmid is designed for expression in E. coli.
Plasmid #10 contains a C-terminal CL7 tag that is downstream of both a PreScission protease (PSC) and Sortase A (SRT) cleavage site. TriAltus scientists find that Sortase A is less efficient than PSC at cleaving membrane proteins.
Plasmid #10 contains two SRT cleavage sites, one downstream of the His8 tag and one upstream of the CL7 tag. The PSC and second SRT cleavage sites are incorporated into an 82-amino acid linker/spacer region downstream of the target insertion site.
An N-terminal His8 tag is located upstream of a SRT cleavage site.