Inspired and stabilized by nature: ribosomal synthesis of the human voltage gated ion channel (VDAC) into 2D-protein-tethered lipid interfaces

Abstract

We present an elegant synthesis and reconstitution approach for functional studies of voltage responsive membrane proteins. For such studies, we propose a planar architecture of an S-layer-supported lipid membrane as a suitable matrix for presenting unmodified membrane protein species, and here we focus on the voltage-dependent anion channel (VDAC) from human mitochondria. The presented cell-free strategy, in which VDAC proteins are synthesized in bacterial cell lysate, into a membrane structure, offers a great advantage in the study of such subtle membrane proteins over the conventional, cell-based synthesis approach in terms of reproducibility. The material-assay combination is superior over cell-culture related synthesis and purification approaches as here we bypass by a one-step synthesis procedure the complex cell culture, and expression and purification endeavours, and, moreover, the protein of interest never has to be detergent solubilized and had been synthesized de novo. We provide here a detailed description from the all over procedure and our first results, describing in detail the cell-free synthesis and robustness of such a material-assay combination: functional VDAC protein species embedded in a planar membrane architecture and ready for electrochemical characterization.

Enhancing the cell-free expression of native membrane proteins by in-silico optimization of the coding sequence – an experimental study of the human voltage-dependent anion channel

“…The production of membrane proteins outside living cells circumvents many of the issues of in-13 cell synthesis. [1,2] Cell-free synthesis uses cell lysates to in situ generate rightly folded 14 membrane proteins [3,4] from exogeneous mRNA or DNA, which can be directly incorporated 15 into artificial membranes. [5] 16…”

Electrochemical Biosensors Based on S-Layer Proteins

“…EIS studies probed the function of both, the membrane-active peptide gramicidin and the membrane protein VDAC. The incorporated VDAC caused a decrease in membrane resistance but the membrane capacitance did not vary significantly [70]. Moreover, the membrane resistance dropped down with increasing VDAC concentration indicating that an increasing number of functional channels spontaneously reconstituted into the SsLM [70].…”

“…EIS can be used to investigate the properties of a material or specific processes that can affect the capacitivity or conductivity/resistivity of an electrochemical system [2,4,68]. EIS technique is widely used in electrochemical biosensors based on S-layer proteins for several applications to assess membrane-active peptide insertion and membrane protein reconstitution into artificial lipid membranes and specific cell detection [69][70][71][72][73].…”

“…SsLMs, generated by the europium-induced vesicle fusion technique [123] provided a proper matrix for the insertion of gramicidin [69] and the voltage-dependent anion channel (VDAC) [70]. EIS studies probed the function of both, the membrane-active peptide gramicidin and the membrane protein VDAC.…”

A cell-free platform for rapid synthesis and testing of active oligosaccharyltransferases

“…Moreover, the open reaction environment allows for precise control of reaction conditions, which is increasingly being applied to the biosynthesis of active membrane proteins. In recent years, a growing number of examples include the manufacture of ATP synthase (Matthies, Haberstock, Joos, Dotsch, & Al., 2011), G-protein-coupled receptors (Corin et al, 2011;Kaiser et al, 2008;Proverbio et al, 2013), and human mitochondrial voltagedependent anion channel (Damiati et al, 2015). The key idea is to synthesize membrane proteins in the presence of natural or synthetic lipids and/or detergents that help solubilize the membrane protein.…”