Activation of biological membrane processes in cell-free reactions

Cell-free biology offers many advantages for the study and utilization of biological membrane processes. As opposed to reductionist biology techniques that examine single component interactions, cell-free biology seeks to activate complex metabolic pathways in an in vitro setting. In previous studies, cell-free biology has been used to gain further understanding of biological membrane processes. Here, we present advances in the activation of cell-free membrane protein synthesis, the development of a membrane isolation method that enables the scale-up of cell-free membrane protein synthesis, and the activation of cell-free lipid synthesis for novel applications.; Membrane proteins have great physiological importance for living organisms and have numerous interesting functions. Aquaporin Z (AqpZ) is a membrane protein that is capable of high water transport rates, while maintaining high selectivity against other molecules. This property makes it a compelling candidate for use in a water filtration device. The realization of such a device would require production of large quantities of aquaporin Z, but current methods for membrane protein production are inadequate. To achieve high quantities of AqpZ production, we demonstrate that two cell-free protein synthesis platforms are capable of producing high quantities of active AqpZ. The first system employs synthetic liposomes to provide the specific environment required for proper AqpZ activation. The second system uses natural inner membranes vesicles to properly fold and activate AqpZ. Both systems are capable of producing approximately 500 mug/mL of membrane inserted AqpZ that exhibits natural water transport activity and selectivity, as characterized by stopped-flow light scattering. Cell-free production of membrane proteins in natural inner membrane vesicles mimics the in vivo process of membrane protein insertion. While this increases the likelihood of successful membrane protein production, one major limitation is an inability to prepare sufficient quantities of inner membrane vesicles for large scale studies. We have developed a method that can be used to facilitate the scalable production of inner membrane vesicles using a biotin-streptavidin affinity interaction. We observed that biotinylated inner membrane vesicles have a high affinity for immobilized streptavidin and can be released without harming vesicle function.; Lipid synthesis is essential for growth and is responsible for maintaining the lipid bilayer that houses membrane proteins. Cell-free production of lipids has many interesting applications including the repair of defects in planar supported lipid bilayers made from natural inner membranes, the production of biofuels using an energy efficient synthesis pathway, and the high-throughput screening of potential lipid synthesis inhibiting antibiotics. For these applications, we have demonstrated the feasibility of cell-free lipid synthesis, identified two factors limiting production, and confirmed production of lipids using thin layer chromatography.