Eukaryotic Cell-free Expression Research Articles

In Vitro Selection of RNA Aptamers Binding to Nanosized DNA for Constructing Artificial Riboswitches.

We here designed an in vitro selection scheme for obtaining an aptamer with which to rationally construct an artificial riboswitch as its component part. In fact, a nanosized DNA-binding aptamer obtained through this scheme allowed us to easily and successfully create eukaryotic riboswitches that upregulate internal ribosome entry site-mediated translation in response to the ligand (nanosized DNA) in wheat germ extract, a eukaryotic cell-free expression system. The induction ratio of the best riboswitch ligand-dose-dependently increased to 21 at 300 μM ligand. This switching efficiency is much higher than that of the same type of riboswitch with a widely used theophylline-binding aptamer, which was in vitro selected without considering its utility for constructing riboswitches. The selection scheme described here would facilitate obtaining various ligand/aptamer pairs suitable for constructing artificial riboswitches, which could serve as elements of synthetic gene circuits in synthetic biology.

Adaption of the Leishmania Cell-Free Expression System to High-Throughput Analysis of Protein Interactions.

In this chapter, we present methods for adapting the eukaryotic cell-free expression system based on Leishmania tarentolae to high-throughput analysis of protein interactions. Specifically, we present a lysate optimization technique that minimizes the amount of unwanted premature termination products while balancing protein expression level and protein aggregation. Finally, we present methods for adapting the Leishmania cell-free system to the AlphaLISA-based protein interaction assay.

Canonical translation-modulating OFF-riboswitches with a single aptamer binding to a small molecule that function in a higher eukaryotic cell-free expression system

We have found that OFF-riboswitches that ligand-dependently downregulate the canonical translation in a higher eukaryotic expression system (wheat germ extract) can be easily created by inserting a single aptamer into the 5′ untranslated region (UTR) of mRNA, even if its ligand is as small as theophylline. The key is the position of the inserted aptamer: the 5′ end (+0 position) is much better than other positions for inhibiting canonical translation with the aptamer-ligand complex. The data showed that ribosome loading is suppressed by a rigid structure in the 5′ end, and this suppression is dependent on the structure’s stability but not on its size. Although this preference of aptamer insertion point contradicts the results in a lower eukaryote, it accords with the fact that the 5′-end structural hindrance is more effective for blocking the ribosome in higher eukaryotes. Therefore, the present type of OFF-riboswitch would function in various higher eukaryotic expression systems.

HERG Ion Channel Activity Measured in Planar Lipid Bilayer Arrays: Reconstitution from Eukaryotic Cell-Free Expression System and Cellular Membrane Preparations

Ion channels are important pharmacological targets. Therefore there is an urgent need for new technologies for high throughput ion channel activity monitoring and pharmacology screening capable to substitute currently used labor-intensive and expensive cell-based electrophysiological assays. Ion channels reconstituted into artificial membrane arrays may serve as such test system offering a substantial simplification as well as great flexibility in controlling the ion channel environment and experimental conditions.Human ether-a-go-go-related gene (hERG), a voltage dependent potassium channel is a major target of pharmacology safety screening due to its critical importance in heart function. Here we report on parallel high-resolution electrophysiological recordings from different hERG-containing preparations reconstituted into bilayer lipid membranes on the micro electrode cavity array (MECA) chip.Two different sources of hERG channels have been investigated: (1) Membrane preparations from a hERG-expressing HEK293 cell line, and (2) Vesicles collected after cell-free expression of the hERG channels with their co-translational integration into microsomes or liposomes. The hERG-containing vesicles were fused with lipid bilayers preformed on the MECA chip. For both preparations fluctuating currents of different amplitudes have been recorded following the fusion. Single channel and channel ensemble currents depending on the vesicle preparation have been characterized. The recorded currents were shown to be partially inhibited by the hERG specific blockers E4031 and astemizole.High channel expression level as well as optimized vesicles preparation and fusion conditions were shown to be crucial for the reproducible channel recordings in the artificial membrane system. Addition of charged lipids (POPG/DOTAP) to the liposomes and acceptor membranes was found to facilitate vesicle fusion and improve the measurements success rate. The presented technology has a potential for a high throughput ion channels pharmacology screening in general.

IRES-Mediated Translation of Membrane Proteins and Glycoproteins in Eukaryotic Cell-Free Systems

Internal ribosome entry site (IRES) elements found in the 5′ untranslated region of mRNAs enable translation initiation in a cap-independent manner, thereby representing an alternative to cap-dependent translation in cell-free protein expression systems. However, IRES function is largely species-dependent so their utility in cell-free systems from different species is rather limited. A promising approach to overcome these limitations would be the use of IRESs that are able to recruit components of the translation initiation apparatus from diverse origins. Here, we present a solution to this technical problem and describe the ability of a number of viral IRESs to direct efficient protein expression in different eukaryotic cell-free expression systems. The IRES from the intergenic region (IGR) of the Cricket paralysis virus (CrPV) genome was shown to function efficiently in four different cell-free systems based on lysates derived from cultured Sf21, CHO and K562 cells as well as wheat germ. Our results suggest that the CrPV IGR IRES-based expression vector is universally applicable for a broad range of eukaryotic cell lysates. Sf21, CHO and K562 cell-free expression systems are particularly promising platforms for the production of glycoproteins and membrane proteins since they contain endogenous microsomes that facilitate the incorporation of membrane-spanning proteins and the formation of post-translational modifications. We demonstrate the use of the CrPV IGR IRES-based expression vector for the enhanced synthesis of various target proteins including the glycoprotein erythropoietin and the membrane proteins heparin-binding EGF-like growth factor receptor as well as epidermal growth factor receptor in the above mentioned eukaryotic cell-free systems. CrPV IGR IRES-mediated translation will facilitate the development of novel eukaryotic cell-free expression platforms as well as the high-yield synthesis of desired proteins in already established systems.

Wheat germ cell-free expression system as a pathway to improve protein yield and solubility for the SSGCID pipeline.

Recombinant expression of proteins of interest in Escherichia coli is an important tool in the determination of protein structure. However, lack of expression and insolubility remain significant challenges to the expression and crystallization of these proteins. The SSGCID program uses a wheat germ cell-free expression system as a rescue pathway for proteins that are either not expressed or insoluble when produced in E. coli. Testing indicates that the system is a valuable tool for these protein targets. Further increases in solubility were obtained by the addition of the NVoy polymer reagent to the reaction mixture. These data indicate that this eukaryotic cell-free expression system has a high success rate and that the addition of specific reagents can increase the yield of soluble protein.