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.
We have used the Expresso T7 Cloning and Expression System for expression and purification of a variety of proteins. Some results of an ongoing large-scale expression study to identify hydrolase enzymes from Fibrobacter succinogenes are presented in Figure 3. Initially, 48 genes were selected for expression trials and cloned into the pETite C-His Vector.
Approximately half of these clones have produced soluble, active hydrolase protein, while in other instances target proteins were expressed in an insoluble form. Five of the genes producing insoluble proteins were re-amplified and cloned into the pETite SUMO vector. When the resulting clones were expressed in HI-Control BL21(DE3) cells, recovery of active protein in the soluble fraction was significantly improved in four of the five cases. Although tag removal was not necessary for hydrolase activity, the tag could be removed efficiently by SUMO protease.
The cloning strategy outlined here makes the Expresso System well-suited to high-throughput cloning and expression studies. The convenience of preprocessed vector, elimination of multiple enzyme treatment and clean-up steps, and improved control over leaky expression should also prove beneficial to researchers.