Protein splicing involves highly specific, self-catalyzed peptide bond cleavage reactions which provide new avenue for protein engineering and protein synthesis. Combining the unique self-catalytic property of the Saccharomyces cerevisiae VMA intein (Sce VMA intein) with affinity chromatography, a novel protein purification methodology has been developed which has the potential to drastically reduce the production cost of industrial enzymes. Utilizing the thio-inducible-N-terminal cleavage activity of the Sce VMA intein, we have been able to obtain highly purified proteins after a single column. However, the expression level of the fusion protein varies with the target protein which can sometimes result in very low yield. This project should solve this expression problem by using the inducible C-terminal cleavage activity of the Sce VMA intein. The target protein will be fused to the C-terminus of the intein allowing the N-terminal sequence of the fusion protein to be optimized for high level of protein expression. This C-terminal cleavage system will first be studied in Escherichia coli and then applied to other heterologous protein expression systems with secretory capability, e.g. Pichia pastoris. The latter can potentially be automated for large- scale industrial productions.
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