Yeast as a Heterologous Expression System and structural characterisation of clinically relevant proteins

ABSTRACT

Using yeast as heterologous expression host, this thesis explores two distinctly This thesis investigates two distinct projects, each focusing on proteins associated with human diseases. The research encompasses proteins located both within cell membranes and those secreted extracellularly: a multi-drug transporter from human fungal pathogens of the genus Candida (Cdr1) in Chapter 1, and a human serine protease involved in blood coagulation, Factor XII (FXII) in Chapter4. Despite their differences origin, size, cellular localization, biophysical characteristics, and functions, these proteins are used as case studies to demonstrate the versatility of yeast as a heterologous expression system and their accompanying challenges. This thesis employs both Saccharomyces cerevisiae and Pichia pastoris (formerly Komagataella phaffii) as heterologous expression hosts. Despite the efficacy of topical remedies for less severe instances, unmanaged invasive candidiasis can lead to severe complications. Moreover, it is crucial not to underestimate the significance of COVID-19-related candidiasis pathogenesis. However, the increasing drug resistance, facilitated by the overexpression of Candida Drug Resistance Protein 1 (Cdr1) among pathogenic Candida species presents a significant hurdle in candidiasis treatment. Cdr1's ability to efflux diverse hydrophobic compounds, including lipids, steroids, and rhodamine dye molecules, highlights its role as a versatile multi-drug resistance ABC transporter. Understanding the molecular mechanisms of substrate binding and efflux is crucial, especially in the context of resistance to commonly administered azole-type drugs. Cdr1 was successfully expressed using S. cerevisiae. Using single particle analysis of cryogenic electron microscopy (Cryo-EM) revealed structure of substrate bound Cdr1 at atomic resolution range between 3.45 - 3.73 Angstrom. This study combines biophysical assays, computational analysis, and structural insights from homologs and alternative substrate-bound states, providing a thorough mechanistic understanding of Cdr1's multi-drug resistance. Among the widely used anticoagulants for treating and preventing thrombosis, haemorrhage is a common side effect. Any therapy for thrombotic disorders must carefully balance these risks with potential benefits, considering the severity of side effects. Therefore, there is an increasing demand for effective anticoagulants that do not compromise haemostatic function. The second project's target focuses on Human coagulation factor XII (FXII), a serine protease critical to the intrinsic coagulation pathway, is a promising target for inhibitors due to its minimal or absent risk of causing haemorrhage. Structural validation and characterization of the binding site can offer valuable insights for enhancing drug efficacy. A number of combinations of regulatory elements were tested in effort to improve its expression and secretion using S, cerevisiae. Particularly for the active form of serine protease domain (FXIIa-SPD). P. pastoris showed a successful secretion of FXIIa-SPD at ~ 26kDa using bioreactor fed-batch method.