The ultra-high-affinity results in superior properties versus other tag-based systems.
The protein is expressed in a CL7-tagged vector of your choice (see below for a variety of vectors with C- or N-terminal CL7 tag and additional tags, e.g. trx1 for improved protein folding, or signal peptides for membrane protein expression). The E. coli lysate is applied to the lm7 resin which is available activated in a slurry format or pre-loaded in gravity resp. FPLC columns (see below) and consists of agarose beads which are covalently linked to the Im7 ligand.
2 & 3. Cleave and Collect
The CL7-tagged expression vectors include cleavage sites to remove the CL7 tag (and other tags if applicable) by either Prescission Protease or SUMO Protease. After on-column protease cleavage the tag free clean protein is eluted from the column.
4. Regenerate The lm7 resin can be regenerated with Guanidinium HCl and re-used up to 100 times.
The TriAltus CL7/lm7 system has been used successfully for many different proteins including difficult-to-purify ones such as membrane proteins, DNA/RNA-binding multi-subunit proteins and proteins toxic to E. coli.
Membrane Proteins: Coomassie stained gels comparing His-trap and CL7/Im7 purification of YidC and CNX:
Multi-Subunit, DNA-Binding RNAPs: Coomassie stained gels comparing His-trap and CL7/Im7 purification of ttRNAP and showing one-step Im7 purification of mtRNAP:
Toxic Proteins: Proteins which are toxic to E. coli cannot be over-expressed in the host by nature. The sensitivity of the CL7/lm7 system enables the purification of such difficult proteins at excellent purity, as shown here:
The highly toxic bacterial MukBEF condensin (~250 kDa, 3 subunits) was successfully purified by one step CL7/Im7 purification.
Biosimilar Proteins: TriAltus´ CL7/lm7 system has been extensively used to purify human therapeutically relevant proteins in large quantities and high purities from E. coli. See Coomassie stained gels of human NEF, Growth Hormone, IFNa, GCSF, and Fc, all highly purified with the TriAltus system:
CAS9 Proteins: Purity of Cas9 highly impacts CRISPR-based genome editing using the RNP approach. The CL7/lm7 system enables the one step purification of highly pure and active Cas9 in contrast to the traditionally used his tag purification: