Synthesis of mono Cytochrome P450 in a modified CHO-CPR cell-free protein production platform.

Cytochromes P450 (CYPs) are a group of monooxygenases that can be found in almost all kinds of organisms. For CYPs to receive electrons from co-substrate NADPH, the activity of NADPH-Cytochrome-P450-oxidoreductase (CPR) is required as well. In humans, CYPs are an integral part of liver-based phase-1 biotransformation, which is essential for the metabolization of multiple xenobiotics and drugs. Consequently, CYPs are important players during drug development and therefore these enzymes are implemented in diverse screening applications. For these applications it is usually advantageous to use mono CYP microsomes containing only the CYP of interest. The generation of mono-CYP containing mammalian cells and vesicles is difficult since endogenous CYPs are present in many cell types that contain the necessary co-factors. By obtaining translationally active lysates from a modified CHO-CPR cell line, it is now possible to generate mono CYPs in a cell-free protein synthesis process in a straightforward manner. As a proof of principle, the synthesis of active human CYPs from three different CYP450 gene families (CYP1A2, CYP2B6 and CYP3A4), which are of outstanding interest in industry and academia was demonstrated. Luciferase based activity assays confirm the activity of the produced CYPs and enable the individual adaptation of the synthesis process for efficient cell-free enzyme production. Furthermore, they allow for substrate and inhibitor screenings not only for wild-type CYPs but also for mutants and further CYP isoforms and variants. As an example, the turnover of selected CYP substrates by cell-free synthesized CYPs was demonstrated via an indirect luciferase assay-based screening setup.

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. Here we demonstrate the applicability of the eukaryotic cell-free systems based on lysates from the mammalian Chinese Hamster Ovary (CHO) and insect Spodoptera frugiperda (Sf21) cells. We demonstrate the efficiency of the systems in the de novo cell-free synthesis of the human cardiac ion channels: ether-a-go-go potassium channel (hERG) KV11.1 and the voltage-gated sodium channel hNaV1.5.

Synthesis of an Anti-CD7 Recombinant Immunotoxin Based on PE24 in CHO and E. coli Cell-Free Systems.

Recombinant immunotoxins (RITs) are an effective class of agents for targeted therapy in cancer treatment. In this article, we demonstrate the straight-forward production and testing of an anti-CD7 RIT based on PE24 in a prokaryotic and a eukaryotic cell-free system. The prokaryotic cell-free system was derived from Escherichia coli BL21 StarTM (DE3) cells transformed with a plasmid encoding the chaperones groEL/groES. The eukaryotic cell-free system was prepared from Chinese hamster ovary (CHO) cells that leave intact endoplasmic reticulum-derived microsomes in the cell-free reaction mix from which the RIT was extracted. The investigated RIT was built by fusing an anti-CD7 single-chain variable fragment (scFv) with the toxin domain PE24, a shortened variant of Pseudomonas Exotoxin A. The RIT was produced in both cell-free systems and tested for antigen binding against CD7 and cell killing on CD7-positive Jurkat, HSB-2, and ALL-SIL cells. CD7-positive cells were effectively killed by the anti-CD7 scFv-PE24 RIT with an IC50 value of 15 pM to 40 pM for CHO and 42 pM to 156 pM for E. coli cell-free-produced RIT. CD7-negative Raji cells were unaffected by the RIT. Toxin and antibody domain alone did not show cytotoxic effects on either CD7-positive or CD7-negative cells. To our knowledge, this report describes the production of an active RIT in E. coli and CHO cell-free systems for the first time. We provide the proof-of-concept that cell-free protein synthesis allows for on-demand testing of antibody−toxin conjugate activity in a time-efficient workflow without cell lysis or purification required.

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. Absence or an inadequate amount of carboxylesterase in the endoplasmic reticulum of different eukaryotic cells poses a hindrance to perform calcium imaging in microsomes. In this work, we try to overcome this drawback and adapt the cell-based calcium imaging principle to a cell-free protein synthesis platform. Carboxylesterase synthesized in a Spodoptera frugiperda Sf21 lysate translation system is established as a viable calcium imaging tool in microsomes. Cell-free synthesized carboxylesterase inside microsomes is validated with esterase and dye loading assays. Native proteins from the endoplasmic reticulum, such as ryanodine channels and calcium ATPase, are analysed. Cell-free synthesized transient receptor potential channels are used as model proteins to demonstrate the realization of this concept.

Cell-free synthesis of the hirudin variant 1 of the blood-sucking leech Hirudo medicinalis.

Synthesis and purification of peptide drugs for medical applications is a challenging task. The leech-derived factor hirudin is in clinical use as an alternative to heparin in anticoagulatory therapies. So far, recombinant hirudin is mainly produced in bacterial or yeast expression systems. We describe the successful development and application of an alternative protocol for the synthesis of active hirudin based on a cell-free protein synthesis approach. Three different cell lysates were compared, and the effects of two different signal peptide sequences on the synthesis of mature hirudin were determined. The combination of K562 cell lysates and the endogenous wild-type signal peptide sequence was most effective. Cell-free synthesized hirudin showed a considerably higher anti-thrombin activity compared to recombinant hirudin produced in bacterial cells.

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). Until today, studies have tried to elucidate the structure, complex formation and cell binding mechanisms of the tripartite Nhe toxin. Here, we demonstrate the synthesis of the functional tripartite Nhe toxin using eukaryotic cell-free systems. Single subunits, combinations of two Nhe subunits as well as the complete tripartite toxin were tested. Functional activity was determined by hemolytic activity on sheep blood agar plates, planar lipid bilayer measurements as well as cell viability assessment using the MTT assay. Our results demonstrate that cell-free protein synthesis based on translationally active eukaryotic lysates is a platform technology for the fast and efficient synthesis of functionally active, multicomponent toxins.

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

Cell-free protein synthesis (CFPS) based on eukaryotic Sf21 lysate is gaining interest among researchers due to its ability to handle the synthesis of complex human membrane proteins (MPs). Additionally Sf21 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. Thus microsomal membranes offer a natural environment for de novo synthesized MPs. Despite the advantage of synthesizing complex MPs with post translational modifications directly into the microsomal membranes without any additional solubilization supplements, batch based Sf21 cell-free synthesis suffers from low yields. The bottleneck for MPs in particular after the synthesis and incorporation into the microsomal membranes is to analyze their functionality. Apart from low yields of the synthesized MPs with batch based cell-free synthesis, the challenges arise in the form of cytoskeleton elements and peripheral endogenous proteins surrounding the microsomes which may impede the functional analysis of the synthesized proteins. So careful sample processing after the synthesis is particularly important for developing the appropriate functional assays. Here we demonstrate how MPs (native and batch synthesized) from ER derived microsomes can be processed for functional analysis by electrophysiology and radioactive uptake assay methods. Treatment of the microsomal membranes either with a sucrose washing step in the case of human serotonin transporter (hSERT) and sarco/endoplasmic reticulum Ca2+/ATPase (SERCA) pump or with mild detergents followed by the preparation of proteoliposomes in the case of the human voltage dependent anionic channel (hVDAC1) helps to analyze the functional properties of MPs.

Accelerating the Production of Druggable Targets: Eukaryotic Cell-Free Systems Come into Focus.

In the biopharmaceutical pipeline, protein expression systems are of high importance not only for the production of biotherapeutics but also for the discovery of novel drugs. The vast majority of drug targets are proteins, which need to be characterized and validated prior to the screening of potential hit components and molecules. A broad range of protein expression systems is currently available, mostly based on cellular organisms of prokaryotic and eukaryotic origin. Prokaryotic cell-free systems are often the system of choice for drug target protein production due to the simple generation of expression hosts and low cost of preparation. Limitations in the production of complex mammalian proteins appear due to inefficient protein folding and posttranslational modifications. Alternative protein production systems, so-called eukaryotic cell-free protein synthesis systems based on eukaryotic cell-lysates, close the gap between a fast protein generation system and a high quality of complex mammalian proteins. In this study, we show the production of druggable target proteins in eukaryotic cell-free systems. Functional characterization studies demonstrate the bioactivity of the proteins and underline the potential for eukaryotic cell-free systems to significantly improve drug development pipelines.

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. Herein, we report the cell-free synthesis of human Toll-like receptor 9 (hTLR9) using CHO lysate and the continuous exchange cell-free (CECF) synthesis platform. The functionality of this protein was demonstrated by an ELISA binding assay using the ectodomain of TLR9 (TLR9-ECD).

Cell-free protein synthesis as a novel tool for directed glycoengineering of active erythropoietin.

As one of the most complex post-translational modification, glycosylation is widely involved in cell adhesion, cell proliferation and immune response. Nevertheless glycoproteins with an identical polypeptide backbone mostly differ in their glycosylation patterns. Due to this heterogeneity, the mapping of different glycosylation patterns to their associated function is nearly impossible. In the last years, glycoengineering tools including cell line engineering, chemoenzymatic remodeling and site-specific glycosylation have attracted increasing interest. The therapeutic hormone erythropoietin (EPO) has been investigated in particular by various groups to establish a production process resulting in a defined glycosylation pattern. However commercially available recombinant human EPO shows batch-to-batch variations in its glycoforms. Therefore we present an alternative method for the synthesis of active glycosylated EPO with an engineered O-glycosylation site by combining eukaryotic cell-free protein synthesis and site-directed incorporation of non-canonical amino acids with subsequent chemoselective modifications.

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

The pore forming characteristic of TDH1 and TDH2 variants of thermostable direct hemolysin (TDH), a major toxin involved in the pathogenesis of Vibrio parahaemolyticus, 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. The pore currents were influenced and showed a detectable response in the presence of polymers which makes them suitable for biotechnology applications.

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. Here, we present two cell-free systems derived from Escherichia coli (E. coli) and Spodoptera frugiperda (Sf21) lysates. In the case of the E. coli cell-free system, nanodiscs are used for the solubilization and purification of membrane proteins. In the case of the Sf21 system, endogenous microsomes with an active translocon complex are present within the lysates which facilitate the incorporation of the bacterial potassium channel KcsA within the microsomal membranes. Following cell-free synthesis, these microsomes are directly used for the functional analysis of membrane proteins.

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. We showed significant improvement of protein yield compared to batch formatted reactions and proved biological activity of synthesized proteins using various analysis technologies. Optimized CECF reaction conditions led to membrane protein yields up to 980 µg/ml, which is the highest protein yield reached in a microsome containing eukaryotic cell-free system presented so far.

Cell-free synthesis of functional human epidermal growth factor receptor: Investigation of ligand-independent dimerization in Sf21 microsomal membranes using non-canonical amino acids.

Cell-free protein synthesis systems represent versatile tools for the synthesis and modification of human membrane proteins. In particular, eukaryotic cell-free systems provide a promising platform for their structural and functional characterization. Here, we present the cell-free synthesis of functional human epidermal growth factor receptor and its vIII deletion mutant in a microsome-containing system derived from cultured Sf21 cells. We provide evidence for embedment of cell-free synthesized receptors into microsomal membranes and asparagine-linked glycosylation. Using the cricket paralysis virus internal ribosome entry site and a repetitive synthesis approach enrichment of receptors inside the microsomal fractions was facilitated thereby providing analytical amounts of functional protein. Receptor tyrosine kinase activation was demonstrated by monitoring receptor phosphorylation. Furthermore, an orthogonal cell-free translation system that provides the site-directed incorporation of p-azido-L-phenylalanine is characterized and applied to investigate receptor dimerization in the absence of a ligand by photo-affinity cross-linking. Finally, incorporated azides are used to generate stable covalently linked receptor dimers by strain-promoted cycloaddition using a novel linker system.

Cell-Free Systems Based on CHO Cell Lysates: Optimization Strategies, Synthesis of "Difficult-to-Express" Proteins and Future Perspectives.

Nowadays, biotechnological processes play a pivotal role in target protein production. In this context, Chinese Hamster Ovary (CHO) cells are one of the most prominent cell lines for the expression of recombinant proteins and revealed as a safe host for nearly 40 years. Nevertheless, the major bottleneck of common in vivo protein expression platforms becomes obvious when looking at the production of so called "difficult-to-express" proteins. This class of proteins comprises in particular several ion channels and multipass membrane proteins as well as cytotoxic proteins. To enhance the production of "difficult-to-express" proteins, alternative technologies were developed, mainly based on translationally active cell lysates. These so called "cell-free" protein synthesis systems enable an efficient production of different classes of proteins. Eukaryotic cell-free systems harboring endogenous microsomal structures for the synthesis of functional membrane proteins and posttranslationally modified proteins are of particular interest for future applications. Therefore, we present current developments in cell-free protein synthesis based on translationally active CHO cell extracts, underlining the high potential of this platform. We present novel results highlighting the optimization of protein yields, the synthesis of various "difficult-to-express" proteins and the cotranslational incorporation of non-standard amino acids, which was exemplarily demonstrated by residue specific labeling of the glycoprotein Erythropoietin and the multimeric membrane protein KCSA.

High-yield cell-free synthesis of human EGFR by IRES-mediated protein translation in a continuous exchange cell-free reaction format.

Cell-free protein synthesis systems derived from eukaryotic sources often provide comparatively low amounts of several µg per ml of de novo synthesized membrane protein. In order to overcome this, we herein demonstrate the high-yield cell-free synthesis of the human EGFR in a microsome-containing system derived from cultured Sf21 cells. Yields were increased more than 100-fold to more than 285 µg/ml by combination of IRES-mediated protein translation with a continuous exchange cell-free reaction format that allowed for prolonged reaction lifetimes exceeding 24 hours. In addition, an orthogonal cell-free translation system is presented that enabled the site-directed incorporation of p-Azido-L-phenylalanine by amber suppression. Functionality of cell-free synthesized receptor molecules is demonstrated by investigation of autophosphorylation activity in the absence of ligand and interaction with the cell-free synthesized adapter molecule Grb2.

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. Analytical techniques, including confocal laser scanning microscopy, fluorescence detection of eYFP fusion proteins in a microplate reader and in-gel fluorescence of statistically incorporated fluorescent amino acid derivatives were employed. The reproducibility of our automated synthesis approach is underlined by coefficients of variation below 7.2%. Moreover, the functionality of the cell-free synthesized potassium channel KcsA was analyzed electrophysiologically. Finally, we expanded our cell-free membrane protein synthesis system by an orthogonal tRNA/synthetase pair for the site-directed incorporation of p-Azido-l-phenylalanine based on stop codon suppression. Incorporation was optimized by performance of a two-dimensional screening with different Mg(2+) and lysate concentrations. Subsequently, the selective modification of membrane proteins with incorporated p-Azido-l-phenylalanine was exemplified by Staudinger ligation with a phosphine-based fluorescence dye.

Characterization of trh2 harbouring Vibrio parahaemolyticus strains isolated in Germany.

BACKGROUND: Vibrio parahaemolyticus is a recognized human enteropathogen. Thermostable direct hemolysin (TDH) and TDH-related hemolysin (TRH) as well as the type III secretion system 2 (T3SS2) are considered as major virulence factors. As tdh positive strains are not detected in coastal waters of Germany, we focused on the characterization of trh positive strains, which were isolated from mussels, seawater and patients in Germany. RESULTS: Ten trh harbouring V. parahaemolyticus strains from Germany were compared to twenty-one trh positive strains from other countries. The complete trh sequences revealed clustering into three different types: trh1 and trh2 genes and a pseudogene Ψtrh. All German isolates possessed alleles of the trh2 gene. MLST analysis indicated a close relationship to Norwegian isolates suggesting that these strains belong to the autochthonous microflora of Northern Europe seawaters. Strains carrying the pseudogene Ψtrh were negative for T3SS2ß effector vopC. Transcription of trh and vopC genes was analyzed under different growth conditions. Trh2 gene expression was not altered by bile while trh1 genes were inducible. VopC could be induced by urea in trh2 bearing strains. Most trh1 carrying strains were hemolytic against sheep erythrocytes while all trh2 positive strains did not show any hemolytic activity. TRH variants were synthesized in a prokaryotic cell-free system and their hemolytic activity was analyzed. TRH1 was active against sheep erythrocytes while TRH2 variants were not active at all. CONCLUSION: Our study reveals a high diversity among trh positive V. parahaemolyticus strains. The function of TRH2 hemolysins and the role of the pseudogene Ψtrh as pathogenicity factors are questionable. To assess the pathogenic potential of V. parahaemolyticus strains a differentiation of trh variants and the detection of T3SS2ß components like vopC would improve the V. parahaemolyticus diagnostics and could lead to a refinement of the risk assessment in food analyses and clinical diagnostics.

Cell-free protein synthesis systems derived from cultured mammalian cells.

We present a technology for the production of target proteins using novel cell-free systems derived from cultured human K562 cells and Chinese hamster ovary (CHO) cells. The protocol includes the cultivation of cells, the preparation of translationally active lysates, and the cell-free synthesis of desired proteins. An efficient expression vector based on the internal ribosome entry site (IRES) from the intergenic region (IGR) of the cricket paralysis virus (CrPV) was constructed for both systems. The coupled batch-based platforms enable the synthesis of a broad range of target proteins such as cytosolic proteins, secreted proteins, membrane proteins embedded into endogenous microsomes, and glycoproteins. The glycosylation of erythropoietin demonstrates the successful performance of posttranslational modifications in the novel cell-free systems. Protein yields of approximately 20 µg/ml (K562-based cell-free system) and 50 µg/ml (CHO-based cell-free system) of active firefly luciferase are obtained in the coupled transcription-translation systems within 3 h. As a result, both cell-free protein synthesis systems serve as powerful tools for high-throughput proteomics.