Expression and purification of membrane proteins TCRα and TMPRSS2 for characterization studies

Integral membrane proteins (IMPs) interact with the plasma membrane core, acting as receptors and transporters. They play critical roles in human diseases, as they are involved in signal transduction and are vital for cellular function and normal homeostasis. IMPs account for almost 30% of encoded proteins in all organisms and are targeted by 50% of therapeutic drugs. However, limited structural and functional information is available for most IMPs because they easily aggregate in solution and are challenging to express in quantities suitable for characterization. The expression and purification of IMPs is the first step of characterization studies. Purified proteins are solubilized in membrane mimetics for structural characterization. Most IMPs undergo post-translational modifications that can impact their structure, interaction, and function. An example is N-linked glycosylation, where a sugar moiety is attached to an Asn-X-Ser/Thr residue on an IMP to become an integral membrane glycoprotein. The research strategy described in this dissertation is focused on the recombinant expression of two integral membrane proteins, T-cell receptor alpha (TCRα) and Transmembrane serine protease II (TMPRSS2), which are prospective glycosylation candidates, as they contain the N-linked glycosylation sites and linked to various autoimmune and respiratory diseases in humans. TCRα is an integral part of the T-cell receptor complex responsible for the appropriate immune response to foreign antigens in the cell. TMPRSS2 activates the attachment of the SARS-CoV-2 viral spike protein to the ACE2 receptor, thereby enhancing host cell infection and replication. The recombinant expression and purification of these proteins in bacteria make them readily available for characterization studies to elucidate their structures and functions and determine possible drug candidates for treating diseases associated with them.