Plasmid #6 encodes a mutant PreScission protease (PSCM) that was developed to reduce the self-cleaving activity of the wild-type PreScission protease (PSC). When tested in lysate, this protein was twice as active as the wild-type protease. It has not been tested for on-column cleavage activity.
The sequence for Sortase A protease (SRT) – a Ca2+-dependent protease – is also included in this plasmid.
At a ratio of 1:70 (protease:model protein), 100% of the model protein is cleaved in 15 minutes. Approximately 95% of the model protein is cleaved in 1 hour when the ratio is increased to 1:350.
To increase SRT activity, add 10 mM Tri Gly peptide to the reaction mixture.
Transcription is driven by the T7 RNA polymerase, and the plasmid is designed for expression in E. Coli.
PSCM is produced from Plasmid #6 for isolation via GST, MBP, or His6 purification methods.
Sortase A possesses self-cleaving ability, which releases GST-tagged PSCM from the His6 tag.
An N-terminal MBP region is included at the front of the coding region and a His6 tag is at the end of the coding region.