Electroporation as method for inserting functional membrane proteins in mammalian cells

Gene transfection of a cell, allowing it to express a foreign protein, is a commonly employed tool of cell biology. The result is that the cell acquires a new function, as performed by the protein, and is described as a gain-of-function. Various means of achieving this have been established over the last decades. These methods can broadly be described as stable or transient, depending on the fate of the genetic material. This, in turn, determines the longevity of the protein expression. Genetic material that integrates into the host genome become stably expressed while those that do not will express the protein only as long as the genetic material remains intact in the cell. In either case, the foreign gene is typically controlled by a powerful promoter that ensures a high yield of protein production by the cell. This typically incurs a metabolic load on the cells since they will be driven to divert resources into production of the foreign protein. The result is poor health and even cell death if the metabolic load becomes overwhelming beside the uncontrollability, which is known for therapeutic strategies, based on gene delivery, widely known as gene therapy. An attractive alternative to gene transfection is to introduce the desired membrane protein, ready-made in a suitable membrane fusing architecture, to be inserted directly into the cellular membranes. This effectively circumvents the need for the cells to spend energy to produce target proteins and, moreover, to understand the coding genetic information correctly. Various physical methods have already been developed to do this (the introduction of proteins into cells), including microinjection, osmotic shock and trypsinization. Another relatively easy and fast method is electroporation, which we like to explore as an efficient method for introducing one or even multiple molecules into cells. A distinct advantage of electroporation is the fine control of spatial targeting we like to employ advanced electroporation for tailoring functional membrane proteins, immersed into membrane matrices, into living cells, acting as molecular, intracellular implants. For synthesis of such integrateable membrane protein implants, we have developed a novel strategy, namely the cell-free synthesis of membrane proteins in membrane architectures: phospholipid-based vesicles as well as synthetic membrane analogous: amphiphilic block copolymer architectures. In the presence of a cell-lysate with the necessary ribosomes/chaperones/building and regulating blocks, the respective DNA, coding for the membrane protein of interest, is spontaneously transcribed and finally translated into the membrane protein of interest to be found in the resulting membrane architecture. We have explored this strategy over the last two decades and are optimistic, that this way to synthesize membrane proteins, opens up the path to generate robust and reproducible membrane protein assemblies for characterization as well as for implementation in binding assays in applied research as in the context of drug screening and discovery. Electroporation involves a very short exposure of a cell to an electric field of suitable strength. An excess of exposure or field strength can cause extensive membrane and cell damage leading to cell death, i.e. irreversible electroporation. However, an appropriate strength and duration results in reversible electroporation where a temporary increase of membrane permeability is obtained and cell viability is preserved.

Cells, the building blocks of human organs and tissues, are complex entities which perform thousands of functions at once. The main effectors of such activities, and the most important target of drugs, are membrane proteins. They are embedded into the cell membrane, the protective lipid layer that define the physical entity of a cell. Such proteins are involved in the life cycle, division of cells and communication among cells. Sometimes the cells are not functional due to, for instance, genetic errors in the DNA which lead to the production of a faulty membrane protein. Diseases such as Alzheimer, Parkinson and diabetes are consequences of such genetic errors. Nowadays, besides treating the symptoms of a disease, modern medicine focuses on inserting the correct DNA code for the faulty protein into the cell using a genetic manipulative approach, re-establishing the correct functionality of the membrane protein. However, the bottleneck of such a gene therapeutical approach lies in its lack of controllability and this still represents a fundamental problem: the DNA information cannot be controlled easily in its expression activity resulting in uncontrolled amounts of the protein of interest. In the present project, we opt for a work-around strategy: restoring the function of a faulty membrane protein by in vitro production of a corrected version of such a protein outside the human organism and, subsequently, delivering this membrane protein back into the patient. By this strategy, we thrive on the advantages of gene therapy: correction of diseases at the genetic level. However, with our novel strategy, we benefit from controllable conditions as we perform the correction of a faulty protein outside the human body and deliver only the result the corrected protein version back into the patient. In the present project, we focused on the first crucial step in this strategy: establishing a suitable shuttle system for such corrected, in vitro-synthesized membrane protein. We identified polymeric membranes as optimal (reproducible, robust, non-immunogenic) architectures for hosting membrane proteins. The fusion of such polymersomic shuttle systems carrying membrane proteins with the lipid membrane of a patient's cell was the crucial step in the presented project. We identified electric fields as physical induction of fusion between polymersomes with living cells as a perfect experimental strategy. In the proximity of polymersome membranes we expect the cells to fuse together with the polymersomes once the electric field is deactivated. In this way the protein is transferred from the polymersomes to the membrane of the cell which gains the functionality of the delivered protein. If this process is successful we will have a biomedical methodology to reconstitute the function of a protein in a patient without the application of (uncontrollable) genetic information.