Lucigen currently offers Expresso T7 cloning and expression kits with pETite vectors in three configurations. For routine protein purification by immobilized metal affinity chromatography (IMAC), pETite T7 N-His and pETite T7 C-His Vectors allow fusion to amino-terminal and carboxyl-terminal 6X histidine tags, respectively. Figure 2 shows an example of high-level expression and purification of soluble fluorescent protein with a C-terminal 6xHis tag.
For proteins that are difficult to express in soluble form, the new pETite SUMO vector allows expression of target proteins with an amino-terminal 6xHis-SUMO protein tag.
SUMO (small ubiquitin-like modifier) is a relatively small (100-residue) polypeptide that has been shown to enhance the soluble expression of many proteins that are otherwise difficult to produce in E. coli. After IMAC purification of the N-His-SUMO-tagged protein, the tag portion can be removed precisely by SUMO protease.
The SUMO protease recognizes the tertiary structure of SUMO rather than a short recognition sequence and cleaves precisely at the junction between the SUMO tag and the target protein, with no off-target cleavage. Both the SUMO protease, which is 6xHis tagged, and the cleaved N-His-SUMO tag can then be separated from the released target protein by subtractive IMAC.