(27t) Comparison of GST and SUMO Fusion Tags for Enhanced Yield and Purity of Recombinant Osh4

The Saccharomyces cerevisiae protein Osh4 is an oxysterol binding homologue (Osh) protein involved in non-vesicular lipid trafficking between organelles, and sufficient purified recombinant protein is required to study the mechanism Osh4 uses to bind and traffic lipids. To develop a protocol for purifying Osh4, we evaluated the expression and purification of Osh4 protein using two distinct fusion tags, glutathione S-transferase (GST) and the small ubiquitin-related modifier (SUMO) protein.

Initially, we expressed Osh4 in Escherichia coli BL21(DE3) cells with an N-terminal GST fusion tag that facilitates soluble recombinant protein production and enables purification by affinity chromatography. The soluble E. coli lysate containing Osh4-GST fusion protein was loaded onto a glutathione chromatography column. Subsequently, we employed tobacco etch virus (TEV) protease to cleave Osh4 from the GST tag bound to the column. The Osh4 protein was recovered, and the sample was further purified using size-exclusion chromatography (SEC) to eliminate TEV protease and other impurities. However, this purification method produced 0.03 mg of purified protein per liter of culture which was not enough protein for subsequent experiments, prompting us to explore an alternative expression and purification system.

To address the low yield, we instead expressed Osh4 with an N-terminal 6xHis-SUMO tag. After confirming expression via Western blot, we used immobilized metal affinity chromatography (IMAC) followed by SEC to purify the 6xHis-SUMO-Osh4 fusion protein from the soluble E. coli lysate. SUMO protease was then utilized to cleave Osh4 from the 6xHis-SUMO tag, and the sample was passed through IMAC again to separate the tag and SUMO protease from Osh4 protein. Consequently, we obtained 0.12 mg of purified protein per liter of culture, which is a four-fold increase in the purification yield. A purity of approximately 90% was achieved, which was a substantial improvement compared to the GST fusion construct. Our results show that the SUMO tag enhances the production and purification of recombinant Osh4 protein and highlights the utility of exploring different fusion tags for recombinant protein expression and purification.