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September 15, 2010 (Vol. 30, No. 16)

Implementing an Enzyme-Free Cloning Strategy

System Simplifies Target Gene Cloning and Soluble Protein Expression in E. coli

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    Figure 2. Purification of a 6xHis-tagged fluorescent protein: HI-Control BL21(DE3) cells harboring a yellow fluorescent protein (YFP) gene in the pETite C-His Vector were induced with 1 mM IPTG for 4 hours (lane 2); uninduced cells are shown in lane 1. Cleared cell lysate was loaded onto an Ni-NTA Sepharose® column. Column flow-through (lane 3, FT) and wash (lane 4, W) fractions were collected. The bound YFP was eluted with buffer containing 300 mM imidazole (lanes 5–12, E1–E8). Fluorescent fractions align with the pure recombinant protein as shown on the gel.

    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.

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    Figure 3. Large-scale cloning and expression case study: (A) PCR products from 48 putative hydrolase genes ranging from ~1 to >3 kb. (B) Uninduced (-) and IPTG-induced (+) samples of HI-Control BL21(DE3) cells with 6 different genes cloned into the pETite C-HIS Vector. (C) Enhanced solubility of SUMO-tagged 2201 and 2442 gene products. Total cell extract and soluble fractions are shown. (D) Removal of 6xHis-SUMO tag from purified SUMO-2201 fusion protein by SUMO protease. –prot: uncleaved SUMO-2201 fusion protein after IMAC purification; +prot: SUMO protease-treated fusion protein; C: isolated 2201 protein after removal of 6xHis-SUMO fragment and SUMO protease 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.

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