Plasmid #22 enables the tagging of target proteins with a cleavable CL7 at the N-terminus and an uncleavable His8 at the C-terminus. There are three options for the 5’ placement of target genes (see scheme below), and SpeI is the 3’ cloning site.
Transcription is induced with IPTG and driven by the T7 RNA polymerase. The plasmid is designed for expression in E. coli.
The N-terminus of the protein is tagged with CL7.
SUMO and Sortase A cleavage sites are both located between the CL7 tag and the target protein’s N-terminus.
The coding region begins with a Trx tag and ends with a His8 tag in Plasmid #22.
- HindIII/SpeI Insertion Site – Trx | CL7 | 20-Amino Acid Linker | SUMO | SRT | 13-Amino Acid Linker | Gene of Interest | His8
- KpnI/SpeI Insertion Site – Trx | CL7 | 20-Amino Acid Linker | SUMO | 3| Gene of Interest | His8
- Bsu36I/SpeI Insertion Site – Trx | CL7 | 20-Amino Acid Linker | SUMO | Gene of Interest | His8
The Bsu361/SpeI 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: