Plasmid #10

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. EXPRESSION: Transcription is induced with IPTG and driven by the T7 RNA polymerase. The plasmid is designed for expression in E. coli. PEPTIDE TAG: 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.CLEAVAGE SITE(S): 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. OTHER TAGS: An N-terminal His8 tag is located upstream of a SRT cleavage site. Plasmid guide with plasmid sequence file is available at Trialtusbioscience.com.