A Cost-Effective Cell-Free System Driven by Glycolytic Intermediates Enables the Production of Complex Eukaryotic Proteins.

Cell-free systems are particularly attractive for screening applications and the production of difficult-to-express proteins. However, the production of cell lysates is difficult to implement on a larger scale due to large time requirements, cultivation costs, and the supplementation of cell-free reactions with energy regeneration systems. Consequently, the methylotrophic yeast , which is widely used in recombinant protein production, was utilized in the present study to realize cell-free synthesis in a cost-effective manner.

Unraveling the kinetics and pharmacology of human PepT1 using solid supported membrane-based electrophysiology.

The human Peptide Transporter 1 (hPepT1) is known for its broad substrate specificity and its ability to transport (pro-)drugs. Here, we present an in-depth comprehensive study of hPepT1 and its interactions with various substrates via solid supported membrane-based electrophysiology (SSME). Using hPepT1-containing vesicles, we could not identify any peptide induced pre-steady-state currents, indicating that the recorded peak currents reflect steady-state transport.

Enhancing the performance of a mutant pyrrolysyl-tRNA synthetase to create a highly versatile eukaryotic cell-free protein synthesis tool.

Modification of proteins with a broad range of chemical functionalities enables the investigation of protein structure and activity by manipulating polypeptides at single amino acid resolution. Indeed, various functional groups including bulky non-canonical amino acids like strained cyclooctenes could be introduced by the unique features of the binding pocket of the double mutant pyrrolysyl-tRNA synthetase (Y306A, Y384F), but the instable nature of the enzyme limits its application in vivo. Here, we constructed a cell-free protein production system, which increased the overall enzyme stability by combining different reaction compartments.

Cell-Free Synthesis and Electrophysiological Analysis of Multipass Voltage-Gated Ion Channels Tethered in Microsomal Membranes.

Cell-free protein synthesis (CFPS) has emerged as a powerful tool for the rapid synthesis and analysis of various structurally and functionally distinct proteins. These include 'difficult-to-express' membrane proteins such as large multipass ion channel receptors. Owing to their membrane localization, eukaryotic CFPS supplemented with endoplasmic reticulum (ER)-derived microsomal vesicles has proven to be an efficient system for the synthesis of functional membrane proteins.

Evaluation of the Ion Channel Assembly in a Eukaryotic Cell-Free System Focusing on Two-Pore Domain Potassium Channels K.

Oligomeric ion channels are abundant in nature. However, the recombinant expression in cell culture-based systems remains tedious and challenging due to negative side effects, limiting the understanding of their role in health and disease. Accordingly, in this work, we demonstrate the cell-free synthesis (CFS) as an alternative platform to study the assembly of two-pore domain potassium channels (K) within endogenous endoplasmic reticulum-derived microsomes.

Enriched cell-free and cell-based native membrane derived vesicles (nMV) enabling rapid in-vitro electrophysiological analysis of the voltage-gated sodium channel 1.5.

Here, we demonstrate the utility of native membrane derived vesicles (nMVs) as tools for expeditious electrophysiological analysis of membrane proteins. We used a cell-free (CF) and a cell-based (CB) approach for preparing protein-enriched nMVs. We utilized the Chinese Hamster Ovary (CHO) lysate-based cell-free protein synthesis (CFPS) system to enrich ER-derived microsomes in the lysate with the primary human cardiac voltage-gated sodium channel 1.

The Potential of Eukaryotic Cell-Free Systems as a Rapid Response to Novel Zoonotic Pathogens: Analysis of SARS-CoV-2 Viral Proteins.

The ongoing pandemic caused by the novel coronavirus (SARS-CoV-2) has led to more than 445 million infections and the underlying disease, COVID-19, resulted in more than 6 million deaths worldwide. The scientific world is already predicting future zoonotic diseases. Hence, rapid response systems are needed to tackle future epidemics and pandemics.

Targeted esterase-induced dye (TED) loading supports direct calcium imaging in eukaryotic cell-free systems.

Calcium imaging is an important functional tool for analysing ion channels, transporters and pumps for drug screening in living cells. Depicted eukaryotic cell-free systems utilize microsomes, derived from the endoplasmic reticulum to incorporate the synthesized membrane proteins-like ion channels. Carboxylesterase is required to cleave the acetoxymethyl ester moiety of the chemical calcium indicators in order to ensure its immobility across the endoplasmic reticulum membrane.

SSM-based electrophysiology, a label-free real-time method reveals sugar binding & transport events in SGLT1.

Here, we present a solid-supported membrane (SSM)-based electrophysiological approach to study sugar binding and Na/glucose cotransport by SGLT1 in membrane vesicles. SSM-based electrophysiology delivers a cumulative real-time current readout from numerous SGLT1 proteins simultaneously using a gold-coated sensor chip. In contrast to conventional techniques, which mainly operate with voltage steps, currents are triggered by sugar or sodium addition.

Cell-Free Protein Synthesis: A Promising Option for Future Drug Development.

Proteins are the main source of drug targets and some of them possess therapeutic potential themselves. Among them, membrane proteins constitute approximately 50% of the major drug targets. In the drug discovery pipeline, rapid methods for producing different classes of proteins in a simple manner with high quality are important for structural and functional analysis.

Mammalian cell-free protein expression promotes the functional characterization of the tripartite non-hemolytic enterotoxin from Bacillus cereus.

Bacillus cereus is increasingly recognized as an opportunistic pathogen causing local and systemic infections. The causative strains typically produce three pore-forming enterotoxins. This study focusses on the tripartite non-hemolytic enterotoxin (Nhe).

Functional Reconstitution of Membrane Proteins Derived From Eukaryotic Cell-Free Systems.

Cell-free protein synthesis (CFPS) based on eukaryotic 21 lysate is gaining interest among researchers due to its ability to handle the synthesis of complex human membrane proteins (MPs). Additionally 21 cell-free systems contain endogenous microsomal vesicles originally derived from the endoplasmic reticulum (ER). After CFPS, MPs will be translocated into the microsomal vesicles membranes present in the lysates.

Cell-free synthesis of human toll-like receptor 9 (TLR9): Optimization of synthesis conditions and functional analysis.

The Toll-like receptor family belongs to the group of pathogen recognition receptors which is responsible for the discrimination of self and non-self pathogen-associated molecular patterns (PAMP's). Toll-like receptors play an important role in the innate immunity and defects in protein expression or polymorphism is linked to various diseases such as Systemic Lupus Erythematosus (SLE). The elucidation of the underlying mechanism is crucial for future treatment and therapeutics of toll-like receptor linked diseases.

Cell-free production of pore forming toxins: Functional analysis of thermostable direct hemolysin from .

The pore forming characteristic of TDH1 and TDH2 variants of thermostable direct hemolysin (TDH), a major toxin involved in the pathogenesis of , was studied on a planar lipid bilayer painted over individual picoliter cavities containing microelectrodes assembled in a multiarray. Both proteins formed pores upon insertion into the lipid bilayer which was shown as a shift in the conductance from the baseline current. TDH2 protein was able to produce stable currents and the currents were influenced by external factors like concentration, type of salt and voltage.

Functional Analysis of Membrane Proteins Produced by Cell-Free Translation.

Cell-free production is a valuable and alternative method for the synthesis of membrane proteins. This system offers openness allowing the researchers to modify the reaction conditions without any boundaries. Additionally, the cell-free reactions are scalable from 20 μL up to several mL, faster and suitable for the high-throughput protein production.

High-yield production of "difficult-to-express" proteins in a continuous exchange cell-free system based on CHO cell lysates.

Cell-free protein synthesis (CFPS) represents a promising technology for efficient protein production targeting especially so called "difficult-to-express" proteins whose synthesis is challenging in conventional in vivo protein production platforms. Chinese hamster ovary (CHO) cells are one of the most prominent and safety approved cell lines for industrial protein production. In this study we demonstrated the ability to produce high yields of various protein types including membrane proteins and single chain variable fragments (scFv) in a continuous exchange cell-free (CECF) system based on CHO cell lysate that contains endogenous microsomal structures.

Automated production of functional membrane proteins using eukaryotic cell-free translation systems.

Due to their high abundance and pharmacological relevance there is a growing demand for the efficient production of functional membrane proteins. In this context, cell-free protein synthesis represents a valuable alternative that allows for the high-throughput synthesis of functional membrane proteins. Here, we demonstrate the potential of our cell-free protein synthesis system, based on lysates from cultured Spodoptera frugiperda 21 cells, to produce pro- and eukaryotic membrane proteins with individual topological characteristics in an automated fashion.

Membrane protein synthesis in cell-free systems: from bio-mimetic systems to bio-membranes.

When taking up the gauntlet of studying membrane protein functionality, scientists are provided with a plethora of advantages, which can be exploited for the synthesis of these difficult-to-express proteins by utilizing cell-free protein synthesis systems. Due to their hydrophobicity, membrane proteins have exceptional demands regarding their environment to ensure correct functionality. Thus, the challenge is to find the appropriate hydrophobic support that facilitates proper membrane protein folding.

Membrane assembly of the functional KcsA potassium channel in a vesicle-based eukaryotic cell-free translation system.

The potassium channel KcsA was heterologously expressed in a eukaryotic cell-free system. Both, the expression yields and functional analysis of the protein were reported. Qualitative and quantitative analyses of KcsA expression were performed by using (14)C-labeled leucine as one of the amino acids supplemented in the cell-free reaction mixture.

Label-free biosensing based on single gold nanostars as plasmonic transducers.

Gold nanostars provide high sensitivity for single nanoparticle label-free biosensing. The nanostars present multiple plasmon resonances of which the lower energy ones, corresponding to the nanostar tips and core-tip interactions, are the most sensitive to environmental changes. Streptavidin molecules are detected upon binding to individual, biotin-modified gold nanostars by spectral shifts in the plasmon resonances.

Electrochemical biosensor microarray functionalized by means of biomolecule friendly photolithography.

Microfabrication permits the incorporation of dense electrode arrays in microsystems and small volume diagnostic devices. However, the specific functionalization of arbitrary shape electrodes with different biomolecules remains a challenging issue. In the present work, the problem of fabricating closely spaced microelectrodes (20 microm sensor diameter and 20 microm-spaced interdigitated electrodes array) that can be modified selectively in order to create multi-analyte sensor arrays is addressed by employing a biomolecule friendly photolithographic procedure for the sequential immobilization of different biomolecules onto separated electrodes of the same array.

Label free optical sensor for Avidin based on single gold nanoparticles functionalized with aptamers.

Optical spectroscopy of a single gold nanoparticle, functionalized with an aptamer, is used to sense the specific binding of avidin. Herewith, the field of single noble metal nanoparticle biosensors is extended to the important field of aptamer based assays. The sensitivity of this initial, but not yet optimized apta-nano-sensor is in the range of 20 nM.

Controlled electrophoretic deposition of multifunctional nanomodules for bioelectrochemical applications.

The design of an electrochemical glucose sensing device formed by the electrodeposition of multifunctional Au nanoparticles is reported here as a novel concept for an enhanced generic sensing platform. Initially gold nanoparticles (Au) were alternatively coated with a layer of positively charged redox polymer (ORP) and a negatively charged glucose oxidase (GOX) layer alternatively using layer-by-layer methodology to form multifunctional Au/ORP/GOX/ORP particles. The modification and stability of the Au nanoparticles was monitored by using UV-vis spectroscopy and zeta-potential measurements.