Abstract
Membrane proteins are involved in many aspects of cellular biology; for example, they regulate how cells interact with their environment, so such proteins are important drug targets. The rapid advancement in the field of immune effector cell therapy has been expanding the horizons of synthetic membrane receptors in the areas of cell-based immunotherapy and cellular medicine. However, the investigation of membrane proteins, which are key constituents of cells, is hampered by the difficulty and complexity of their in vitro synthesis, which is of unpredictable yield. Cell-free synthesis is herein employed to unravel the impact of the expression construct on gene transcription and translation, without the complex regulatory mechanisms of cellular systems. Through the systematic design of plasmids in the immediacy of the start of the target gene, it was possible to identify translation initiation and the conformation of mRNA as the main factors governing the cell-free expression efficiency of the human voltage-dependent anion channel (VDAC), which is a relevant membrane protein in drug-based therapy. A simple translation initiation model was developed to quantitatively assess the expression potential for the designed constructs. A scoring function that quantifies the feasibility of the formation of the translation initiation complex through the ribosome–mRNA hybridization energy and the accessibility of the mRNA segment binding to the ribosome is proposed. The scoring function enables one to optimize plasmid sequences and semi-quantitatively predict protein expression efficiencies. This scoring function is publicly available as webservice XenoExpressO at University of Vienna, Austria.
Highlights
Understanding the structure and function of membrane proteins is key in many biological processes yet faces numerous issues
By the introduction of various self-designed primers, we were able to modify the genetic code in a controlled fashion and assess the effect of these modifications on protein expression
The current study demonstrates that the prokaryotic cell-free expression of human voltage-dependent anion channel (VDAC) is determined by the mRNA sequence in the immediacy of the start codon and its impact on translation initiation
Summary
Understanding the structure and function of membrane proteins is key in many biological processes yet faces numerous issues. Membrane proteins are notoriously difficult to synthesize: in cells, membrane proteins are usually expressed in low amounts, and their expression profile is heavily controlled as part of regulatory processes. The characterization of membrane proteins is no less difficult: the structural integrity of membrane proteins is hard to preserve in extracellular conditions, and function may be lost if proteins are removed from their native membranes. The production of membrane proteins outside living cells circumvents many of the issues of in-cell synthesis [1,2]. The functionalization of nanodiscs with VDAC receptor via cell-free protein synthesis was shown recently and in very detailed analysis [8], supporting the in-principle functionality of cell-free in vitro synthesized membrane proteins
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