Cell-Free Production of Human Solute Carriers

The cell membrane forms the barrier separating the interior and the exterior of cells. Embedded into a lipid matrix, membrane proteins (MPs) mediate signal transduction and transport processes. In humans, the solute carriers (SLCs), crucial for the transport of a variety of substrates, as amino acids, neurotransmitters or fatty acids are the second largest family of membrane proteins. Many of them are involved in diseases like cancer and Alzheimer's disease [1], [2]. Although one can find a number of structures for prokaryotic homologs of solute carriers, little is known about human SLCs making them interesting targets for functional and structural studies. SLCs and in general most membrane proteins are present in cells at low concentrations only, making heterologous expression systems to produce sufficient amounts in stable, pure and homogeneous form the methods of choice. Although heterologous production in Escherichia coli or in different eukaryotic cells is commonly employed for the production of recombinant membrane proteins, the in vivo production has several bottlenecks, like the impossibility to produce toxic proteins, proteolytic degradation or the formation of inclusion bodies. Cell-free expression (CF) is an emerging technique to circumvent many key problems of MP expression in host cells. Toxic or growth inhibitory effects can be eliminated and several additives (e.g. detergents, chaperones) can be provided during the reaction to stabilize the nascent protein [3]. For CF MP production almost solely S30 extracts from E. coli have been used until now [4]. Albeit the E. coli system is robust and reliable, its source is nevertheless prokaryotic. Due to the prokaryotic nature of the translation machinery, proteins from higher organisms tend to missfold in the E. coli system [5]. This observation and the fact that the E. coli cell-free system suffers from the absence of eukaryotic-specific post-translational modifications, makes the establishment of eukaryotic cell-free expression systems necessary. Based on the protozoan Leishmania tarentolae, a cell-free system could be established recently in parallel to the system published by Mureev et al. [6]. This system could be used successfully to express a number of human SLCs from different families. It was possible to solubilize selected transporters and to confirm their identity via mass spectrometry. Purification and further characterization are underway.