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 1.5 (hNaV1.5; SCN5A) in 3 h. Subsequently, CB-nMVs were isolated from fractions of nitrogen-cavitated CHO cells overexpressing the hNaV1.5. In an integrative approach, nMVs were micro-transplanted into Xenopus laevis oocytes. CB-nMVs expressed native lidocaine-sensitive hNaV1.5 currents within 24 h; CF-nMVs did not elicit any response. Both the CB- and CF-nMV preparations evoked single-channel activity on the planar lipid bilayer while retaining sensitivity to lidocaine application. Our findings suggest a high usability of the quick-synthesis CF-nMVs and maintenance-free CB-nMVs as ready-to-use tools for in-vitro analysis of electrogenic membrane proteins and large, voltage-gated ion channels

A Cost-Effective Pichia pastoris 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 Pichia pastoris, which is widely used in recombinant protein production, was utilized in the present study to realize cell-free synthesis in a cost-effective manner. Sensitive disruption conditions were evaluated, and appropriate signal sequences for translocation into ER vesicles were identified. An alternative energy regeneration system based on fructose-1,6-bisphosphate was developed and a ~2-fold increase in protein production was observed. Using a statistical experiment design, the optimal composition of the cell-free reaction milieu was determined. Moreover, functional ion channels could be produced, and a G-protein-coupled receptor was site-specifically modified using the novel cell-free system. Finally, the established P. pastoris cell-free protein production system can economically produce complex proteins for biotechnological applications in a short time

Stable Chinese Hamster Ovary Suspension Cell Lines Harboring Recombinant Human Cytochrome P450 Oxidoreductase and Human Cytochrome P450 Monooxygenases as Platform for In Vitro Biotransformation Studies

In the liver, phase-1 biotransformation of drugs and other xenobiotics is largely facilitated by enzyme complexes consisting of cytochrome P450 oxidoreductase (CPR) and cytochrome P450 monooxygenases (CYPs). Generated from human liver-derived cell lines, recombinant in vitro cell systems with overexpression of defined phase-1 enzymes are widely used for pharmacological and toxicological drug assessment and laboratory-scale production of drug-specific reference metabolites. Most, if not all, of these cell lines, however, display some background activity of several CYPs, making it difficult to attribute effects to defined CYPs. The aim of this study was to generate cell lines with stable overexpression of human phase-1 enzymes based on Chinese hamster ovary (CHO) suspension cells. Cells were sequentially modified with cDNAs for human CPR in combination with CYP1A2, CYP2B6, or CYP3A4, using lentiviral gene transfer. In parallel, CYP-overexpressing cell lines without recombinant CPR were generated. Successful recombinant expression was demonstrated by mRNA and protein analyses. Using prototypical CYP-substrates, generated cell lines proved to display specific enzyme activities of each overexpressed CYP while we did not find any endogenous activity of those CYPs in parental CHO cells. Interestingly, cell lines revealed some evidence that the dependence of CYP activity on CPR could vary between CYPs. This needs to be confirmed in further studies. Recombinant expression of CPR was also shown to enhance CYP3A4-independent metabolisation of testosterone to androstenedione in CHO cells. We propose the novel serum-free CHO suspension cell lines with enhanced CPR and/or defined CYP activity as a promising “humanised” in vitro model to study the specific effects of those human CYPs. This could be relevant for toxicology and/or pharmacology studies in the pharmaceutical industry or medicine

Vesicle-based cell-free synthesis of short and long unspecific peroxygenases

Unspecific peroxygenases (UPOs, EC 1.11.2.1) are fungal enzymes that catalyze the oxyfunctionalization of non-activated hydrocarbons, making them valuable biocatalysts. Despite the increasing interest in UPOs that has led to the identification of thousands of putative UPO genes, only a few of these have been successfully expressed and characterized. There is currently no universal expression system in place to explore their full potential. Cell-free protein synthesis has proven to be a sophisticated technique for the synthesis of difficult-to-express proteins. In this work, we aimed to establish an insect-based cell-free protein synthesis (CFPS) platform to produce UPOs. CFPS relies on translationally active cell lysates rather than living cells. The system parameters can thus be directly manipulated without having to account for cell viability, thereby making it highly adaptable. The insect-based lysate contains translocationally active, ER-derived vesicles, called microsomes. These microsomes have been shown to allow efficient translocation of proteins into their lumen, promoting post-translational modifications such as disulfide bridge formation and N-glycosylations. In this study the ability of a redox optimized, vesicle-based, eukaryotic CFPS system to synthesize functional UPOs was explored. The influence of different reaction parameters as well as the influence of translocation on enzyme activity was evaluated for a short UPO from Marasmius rotula and a long UPO from Agrocybe aegerita. The capability of the CFPS system described here was demonstrated by the successful synthesis of a novel UPO from Podospora anserina, thus qualifying CFPS as a promising tool for the identification and evaluation of novel UPOs and variants thereof

Evaluation of the Ion Channel Assembly in a Eukaryotic Cell-Free System Focusing on Two-Pore Domain Potassium Channels K2P

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 (K2P) within endogenous endoplasmic reticulum-derived microsomes. Exploiting the open nature of CFS, we investigate the cotranslational translocation of TREK-2 into the microsomes and suggest a cotranslational assembly with typical single-channel behavior in planar lipid-bilayer electrophysiology. The heteromeric assembly of K2P channels is a contentious matter, accordingly we prove the successful assembly of TREK-2 with TWIK-1 using a biomolecular fluorescence complementation assay, Western blot analysis and autoradiography. The results demonstrate that TREK-2 homodimer assembly is the initial step, followed by heterodimer formation with the nascent TWIK-1, providing evidence of the intergroup heterodimerization of TREK-2 and TWIK-1 in eukaryotic CFS. Since K2P channels are involved in various pathophysiological conditions, including pain and nociception, CFS paves the way for in-depth functional studies and related pharmacological interventions. This study highlights the versatility of the eukaryotic CFS platform for investigating ion channel assembly in a native-like environment

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. Moreover, a co-expression approach in a one-pot reaction allowed straightforward site-specific fluorescent labeling of the functional complex membrane protein cystic fibrosis transmembrane conductance regulator. Our work provides a versatile platform for introducing various non-canonical amino acids into difficult-to-express proteins for structural and fluorescence based investigation of proteins activity

Rapid One-Step Capturing of Native, Cell-Free Synthesized and Membrane-Embedded GLP-1R

G protein-coupled receptors (GPCRs) are of outstanding pharmacological interest as they are abundant in cell membranes where they perform diverse functions that are closely related to the vitality of cells. The analysis of GPCRs in natural membranes is laborious, as established methods are almost exclusively cell culture-based and only a few methods for immobilization in a natural membrane outside the cell are known. Within this study, we present a one-step, fast and robust immobilization strategy of the GPCR glucagon-like peptide 1 receptor (GLP-1R). GLP-1R was synthesized in eukaryotic lysates harboring endogenous endoplasmic reticulum-derived microsomes enabling the embedment of GLP-1R in a natural membrane. Interestingly, we found that these microsomes spontaneously adsorbed to magnetic Neutravidin beads thus providing immobilized membrane protein preparations which required no additional manipulation of the target receptor or its supporting membrane. The accessibility of the extracellular domain of membrane-embedded and bead-immobilized GLP-1R was demonstrated by bead-based enzyme-linked immunosorbent assay (ELISA) using GLP-1R-specific monoclonal antibodies. In addition, ligand binding of immobilized GLP-1R was verified in a radioligand binding assay. In summary, we present an easy and straightforward synthesis and immobilization methodology of an active GPCR which can be beneficial for studying membrane proteins in general

Promoting the production of challenging proteins via induced expression in CHO cells and modified cell-free lysates harboring T7 RNA polymerase and mutant eIF2α

Chinese hamster ovary (CHO) cells are crucial in biopharmaceutical production due to their scalability and capacity for human-like post-translational modifications. However, toxic proteins and membrane proteins are often difficult-to-express in living cells. Alternatively, cell-free protein synthesis can be employed. This study explores innovative strategies for enhancing the production of challenging proteins through the modification of CHO cells by investigating both, cell-based and cell-free approaches. A major result in our study involves the integration of a mutant eIF2 translation initiation factor and T7 RNA polymerase into CHO cell lysates for cell-free protein synthesis. This resulted in elevated yields, while eliminating the necessity for exogenous additions during cell-free production, thereby substantially enhancing efficiency. Additionally, we explore the potential of the Rosa26 genomic site for the integration of T7 RNA polymerase and cell-based tetracycline-controlled protein expression. These findings provide promising advancements in bioproduction technologies, offering flexibility to switch between cell-free and cell-based protein production as needed

One to one comparison of cell-free synthesized erythropoietin conjugates modified with linear polyglycerol and polyethylene glycol

With more than 20 Food and Drug Administration (FDA)-approved poly (ethylene glycol) (PEG) modified drugs on the market, PEG is the gold standard polymer in bioconjugation. The coupling improves stability, efficiency and can prolong blood circulation time of therapeutic proteins. Even though PEGylation is described as non-toxic and non-immunogenic, reports accumulate with data showing allergic reactions to PEG. Since PEG is not only applied in therapeutics, but can also be found in foods and cosmetics, anti-PEG-antibodies can occur even without a medical treatment. Hypersensitivity to PEG thereby can lead to a reduced drug efficiency, fast blood clearance and in rare cases anaphylactic reactions. Therefore, finding alternatives for PEG is crucial. In this study, we present linear polyglycerol (LPG) for bioconjugation as an alternative polymer to PEG. We report the conjugation of LPG and PEG by click-chemistry to the glycoprotein erythropoietin (EPO), synthesized in a eukaryotic cell-free protein synthesis system. Furthermore, the influence of the polymers on EPOs stability and activity on a growth hormone dependent cell-line was evaluated. The similar characteristics of both bioconjugates show that LPGylation can be a promising alternative to PEGylation

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. Electrogenic co-transport of H+/glycylglycine (GlyGly) was observed across a pH range of 5.0 to 9.0. The pH dependence is described by a bell-shaped activity curve and two pK values. KM and relative Vmax values of various canonical and non-canonical peptide substrates were contextualized with current mechanistic understandings of hPepT1. Finally, specific inhibition was observed for various inhibitors in a high throughput format, and IC50 values are reported. Taken together, these findings contribute to promoting the design and analysis of pharmacologically relevant substances