Abstract by Hajira Ahmed Hotiana
Chemical reactions that are responsible for the provision of energy to biological systems are essential for life. These reactions take place in an intricate network of signalling pathways that are carefully maintained and collectively known as human metabolism. Given the mere complexity of the biological pathways involving proteins, protein analysis remains one of the most complex and challenging fields of study. Setting up efficient and reproducible expression and purification pipelines for different types of proteins is a prerequisite for all such downstream studies. We developed protocols and procedures for the overproduction of several challenging protein targets ranging from soluble proteins to membrane proteins that have evaded essential characterization so far due to difficulties in their recombinant production. On the soluble protein front, we present strategies to optimize construct design, expression conditions and purification strategies to gain yields as high as 35 mg of purified protein per L of E. coli culture for the human ER chaperone glucose-regulated protein 94 (GRP94). Using SEC as a tool we assessed the quality of the produced protein and demonstrate functionality using nano differential scanning fluorimetery (nanoDSF).
We shed light on the role ER resident chaperone GRP94 plays in the folding of an insulin precursor proinsulin. Insulin is a widely used biologic that allows millions of diabetic patients worldwide to lead a healthy, normal lifestyle. With the drastic increase in the demand for insulin, the commercial supply of insulin needs to be rapidly improved. We present the first study to understand the molecular mechanism of how human GRP94 interacts specifically with unfolded proinsulin, the binding interface and the structural basis for this interaction to occur. We demonstrate the molecular mechanism of interaction between proinsulin and GRP94 that is essential for proper proinsulin folding. The specificity and mode of action for the interaction between GRP94 and proinsulin outlined in this part of the thesis can have a major biotechnological advantage especially in commercial production of insulin using Saccharomyces cerevisiae that endogenously does not express GRP94. Additionally, we present the structural characteristics of the binding pocket in GRP94 where proinsulin binds and conclude that for the interaction to occur an open conformation of the chaperone must be present. These studies provide the framework to understand how the human β cells cater to proinsulin misfolding and can be extended to develop strategies to prevent the same during commercial insulin production.
This PhD work also includes the study to expand the use of Saccharomyces cerevisiae as a model system for the production of an under characterized membrane protein family, the monocarboxylate transporters (MCTs) that are heavily involved with the import and export of metabolites and metabolic waste. Despite years of interest in MCTs as drug targets, the structural information available for these transporters has been very limited, hindering the development of effective therapeutics against the wide variety of diseases they incur. Membrane proteins are challenging to express and produce especially in sufficient quality and quantity for downstream characterization. With the explosion in the field of cryo-EM as a tool of choice for structural characterization of membrane proteins, new avenues have opened up for overcoming several challenges but protein production for MCTs has still relied on expensive and complicated insect and mammalian cells as host systems. We report the development of a robust and effective set up for the overproduction of MCTs in an easy to genetically manipulate and cheaper Saccharomyces cerevisiae host system, delivering milligrams of homogenous protein samples for downstream characterization. Additionally, we present the successful usage of the transporter protein produced using this platform for cryo-EM studies of human aromatic amino acid transporter, TAT1. This part of the PhD presents an alternative approach for the overproduction of pharmacologically relevant, difficult to express membrane proteins for downstream structural, functional and structure-based drug design studies.