Modern biological research needs simple methods to separate and purify proteins in a single step, with high efficiency and purity. Traditional methods using affinity tags rely on special materials and costly enzymes to remove the tags, making them expensive. To solve these issues, scientists have developed self-cleaving tags that group together (aggregating tags) for protein purification. However, these methods often use inteins (protein segments) that cut at the C-terminal end, but they sometimes cut too early, causing product loss during protein production.
In this study, the researchers tested two new versions (mutants) of the Mtu RecA ΔI-CM mini-intein, created using yeast surface display, to improve protein purification. When combined with an elastin-like-polypeptide (ELP) precipitation tag, the new mutants, called ΔI-12 and ΔI-29, provided better results than the original mini-intein. They produced higher amounts of precursor proteins, better product purity, and improved overall yield. In a single step, these mutants achieved protein purities between 68% and 94% for three test proteins: green fluorescent protein (GFP), maltose-binding protein (MBP), and beta-galactosidase (beta-gal). Additionally, the inteins efficiently cleaved the proteins after just 5 hours under most conditions.
In summary, the researchers developed better self-cleaving tags that can purify a wide range of proteins affordably and effectively at a laboratory scale.
Bron: www.sciencedirect.com