Plasmid #3 serves as a template for tagging target proteins with N-terminal CL7 tag. It has three options for the 5’ placement of target genes (see scheme below), and XhoI is the 3’ cloning site. All three options require a stop codon encoded in the 3’ cloning primer. There is an existing 1711 base pair restriction fragment inserted at the HindIII/XhoI restriction sites.
Transcription is induced with IPTG and driven by the T7 RNA polymerase. The plasmid is designed for expression in E. Coli.
The CL7 tag for purification is fused to the N-terminus of the target protein, upstream of the SUMO cleavage site.
Plasmid #3 includes two protease sites: a SUMO and Sortase A (SRT) cleavage site exist between the CL7 binding site and downstream target protein.
The plasmid includes an N-terminal thioredoxin (Trx) tag upstream of the CL7 tag.
- HindIII/XhoI Insertion Site – Trx | CL7 | SUMO | SRT | Gene of Interest
- KpnI/XhoI Insertion Site – Trx | CL7 | SUMO | Gene of Interest
- Bsu36I/XhoI Insertion Site – Trx | CL7 | SUMO | Gene of Interest
The Bsu36I/XhoI insertion scheme maintains the Gene of Interest’s wildtype sequence without adding any extra residues. The N-terminus of the Gene of Interest must include the following to complete the SUMO C-terminal sequence: