Engineering Platforms for Mimicking Cellular Processes using Cell-Free Expression Systems

Abstract

Bottom-up in vitro reconstitution has provided fundamental insights into the mechanisms of key cellular processes. Traditional reconstitution methods rely on protein purification as the primary means of extracting functional proteins from cells. While efficient for most cytoplasmic proteins which are stable in solution, reconstitution of membrane proteins remains challenging. This is attributed to the requirement of a cellular membrane-like substrate for proper folding and functioning of membrane proteins. Reconstitution into lipid membranes is usually achieved using non-ionic detergents. Such a process is non-trivial and requires excessive optimization based on the type of membrane protein that is to be reconstituted. Additionally, generation of recombinant proteins can hinder protein function post reconstitution. An alternative approach is the use of cell-free expression (CFE) systems to synthesize proteins in vitro. Recent advances in cell-free reconstitution have enabled synthesis and insertion of membrane proteins into lipid bilayer membranes. However, past studies have been primarily focused on the detergent-free reconstitution of membrane proteins without an attempt to recapitulate biologically relevant phenomena. In this dissertation, we demonstrate the potential of using CFE systems to directly investigate important biological processes and to mimic cell-like behavior through the creation of synthetic cells. First, we use a mammalian CFE to study inner nuclear membrane proteins SUN1 and SUN2 that are involved in the formation of the linker of nucleoskeleton and cytoskeleton complex (LINC). Inner nuclear membrane proteins are difficult to study in cells and require sophisticated techniques for successful extraction and analysis. By coupling simple biochemical assays with fluorescence microscopy, we determine the membrane-inserted topology of full-length SUN1 and SUN2 and demonstrate their function in vitro. Further, we find evidence of previously unidentified SUN1-SUN2 heteromeric interactions and possible cation dependent enhancement in binding affinity to KASH peptides which are responsible for the formation of LINC complex in cells. Next, we use an E. coli CFE system encapsulated within lipid bilayer vesicles (liposomes) to demonstrate simultaneous reconstitution of two functionally different proteins. The activity of a CFE reconstituted mechanosensitive channel (MscL) in response to hypo-osmotic shock is demonstrated using a dye leakage assay in liposomes. A calcium biosensor (G-GECO) with high ON/OFF ratio in fluorescence intensity upon calcium binding is successfully expressed using CFE. We observe spontaneous localization of MscL to lipid membranes while G-GECO is homogenously distributed within encapsulated liposomes. Co-expression inside liposomes with application of hypo-osmotic shock result in detectable calcium influx through activated MscL. Our findings present the creation of a synthetic cell which can be used as a reconstitution platform to study calcium permeable mechanosensitive channels. Finally, we develop gene circuits to control the initiation of protein synthesis with inducer molecules in the E. coli CFE. Using a lac promotor to synthesize a green fluorescent protein (deGFP), we demonstrate successful induction with Isopropyl β-d-1-thiogalactopyranoside (IPTG). MscL is reconstituted in liposomes to mediate influx of IPTG in response to hypo-osmotic shock. A high ON/OFF ratio of deGFP synthesis post induction is observed when using a gene amplification circuit. A bacterial cytoskeletal protein MreB substituted in place of deGFP is synthesized inside liposomes in response to hypo-osmotic shock. MreB is observed to form ring-like structures on the liposome membrane. Such a system enables control over initiation of protein synthesis in synthetic cells in response to osmotic changes which is important for the study of spatio-temporal evolution of protein functions.