Skip to main content
Login | Suomeksi | På svenska | In English

Browsing by Subject "Protein purification"

Sort by: Order: Results:

  • Nordlin, Patric (2023)
    The discovery and development of new antifungal drugs has lagged behind the clinical needs for effective treatments of fungal infections. Invasive fungal infections can be challenging to treat and can become life-threatening, particularly for immunosuppressed individuals. Despite the need for new and improved antifungal drugs, the pipeline for antifungal drug development has been relatively slow, with only a few new agents being approved in recent years. Many existing antifungal drugs have toxic side effects, limiting their use and highlighting the need for more targeted and effective therapeutics. The glycosylphosphatidylinositol biosynthesis pathway has been proposed as a potential new target for antifungal drugs. The glycosylphosphatidylinositol (GPI) anchor is a complex glycoconjugate that is attached to many proteins found on the surface of eukaryotic cells. GPI anchored proteins play important roles in various cellular processes, including signaling, cell adhesion, and cell recognition. The biosynthesis of GPI anchors involves multiple enzymatic steps, including the transfer of the GPI anchor to a target protein. Gpi3 is one of the key enzymes involved in the first step of GPI biosynthesis and is the catalytic subunit of the GPI GlcNAc-PI synthesis complex. The naturally occurring molecule Jawsamycin has been shown to selectively inhibit fungal Gpi3 while not interfering with its human ortholog. However, the development and research of Jawsamycin and other potential inhibitors of the GPI synthesis pathway are hampered by the lack of structural data on the proteins involved in the pathway. This thesis aimed to express and purify functionally active Gpi3 as a recombinant fusion protein using the SUMO tag expression system, with the end goal of utilizing the protein for structural studies through crystallography to better understand the function of Jawsamycin. In this thesis, Gpi3 was successfully expressed and purified as a fusion protein. However, enzymatic activity of Gpi3 was not observed, additionally, the purification and stability of the Gpi3 fusion proteins were shown to be problematic and no crystal structure of the protein of interest was acquired. These results indicate that a different approach is needed to gain structural insights into the function and interaction between Gpi3 and Jawsamycin. A likely path forward is the purification of the whole GPI GlcNAc synthesis complex which could give more insights into the organization and function of both Gpi3 and Jawsamycin.