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

Internal ribosome entry site (IRES) elements found in the 5′ untranslated region of mRNAs enable translation initiation a cap-independent manner, thereby representing an alternative to cap-dependent cell-free protein expression systems. However, IRES function is largely species-dependent so their utility systems from different species rather limited. A promising approach overcome these limitations would be use IRESs that are able recruit components apparatus diverse origins. Here, we present...

Abstract Antibodies are indispensable tools for basic research as well diagnostic and therapeutic applications. Consequently, the development of alternative manufacturing strategies which circumvent hurdles connected to conventional antibody production technologies is enormous interest. To address this issue, we demonstrate synthesis complex formats, in particular immunoglobulin G (IgG) single-chain variable fragment Fc fusion (scFv-Fc), a microsome-containing cell-free system based on...

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 expression recombinant proteins and revealed as safe host nearly 40 years. Nevertheless, major bottleneck common vivo platforms becomes obvious when looking at production so called "difficult-to-express" proteins. This class comprises particular several ion channels multipass membrane well cytotoxic To enhance...

In this study, we present a novel technique for the synthesis of complex prokaryotic and eukaryotic proteins by using continuous-exchange cell-free (CECF) protein system based on extracts from cultured insect cells. Our approach consists two basic elements: First, is performed in cell lysates which harbor endogenous microsomal vesicles, enabling translocation de novo synthesized target into lumen vesicles or, case membrane proteins, their embedding natural scaffold. Second, reactions are...

Open cell-free translation systems based on Escherichia coli cell lysates have successfully been used to produce antibodies and antibody fragments. In this study, we demonstrate the expression of functional single-chain variable fragments (scFvs) in a eukaryotic endotoxin-free vitro system Spodoptera frugiperda (Sf21) insect extracts. Three scFv candidates with different specificities were chosen as models. The first candidate SH527-IIA4 specifically discriminates between its phosphorylated...

Cell-free protein synthesis systems derived from eukaryotic sources often provide comparatively low amounts of several μg per ml de novo synthesized membrane protein. In order to overcome this, we herein demonstrate the high-yield cell-free human EGFR in a microsome-containing system cultured Sf21 cells. Yields were increased more than 100-fold 285 μg/ml by combination IRES-mediated translation with continuous exchange reaction format that allowed for prolonged lifetimes exceeding 24 hours....

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 valuable alternative that allows high-throughput Here, we demonstrate potential our system, based on lysates from cultured Spodoptera frugiperda 21 cells, produce pro- eukaryotic proteins with individual topological characteristics in an automated fashion. Analytical techniques, including...

ABSTRACT The biochemical analysis of human cell membrane proteins remains a challenging task due to the difficulties in producing sufficient quantities functional protein. G protein‐coupled receptors (GPCRs) represent main class and drug targets, which are responsible for huge number signaling processes regulating various physiological functions living cells. To circumvent current bottlenecks GPCR studies, we propose synthesis GPCRs eukaryotic cell‐free systems based on extracts generated...

G protein-coupled receptors (GPCRs) are of outstanding pharmacological interest as they abundant in cell membranes where perform diverse functions that closely related to the vitality cells. The analysis GPCRs natural is laborious, established methods almost exclusively culture-based and only a few for immobilization membrane outside known. Within this study, we present one-step, fast robust strategy GPCR glucagon-like peptide 1 receptor (GLP-1R). GLP-1R was synthesized eukaryotic lysates...

Cell-free protein synthesis systems represent versatile tools for the and modification of human membrane proteins. In particular, eukaryotic cell-free provide a promising platform their structural functional characterization. Here, we present epidermal growth factor receptor its vIII deletion mutant in microsome-containing system derived from cultured Sf21 cells. We evidence embedment synthesized receptors into microsomal membranes asparagine-linked glycosylation. Using cricket paralysis...

Abstract 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 structure, complex formation cell binding mechanisms of Nhe toxin. Here, we demonstrate synthesis functional toxin using eukaryotic cell-free systems. Single subunits, combinations two...

Cell-free systems are well-established platforms for the rapid synthesis, screening, engineering and modification of all kinds recombinant proteins ranging from membrane to soluble proteins, enzymes even toxins. Also within antibody field cell-free technology has gained considerable attention with respect clinical research pipeline including discovery production. Besides classical full-length monoclonal antibodies (mAbs), so-called "nanobodies" (Nbs) have come into focus. A Nb is smallest...

The tripartite enterotoxin Hemolysin BL (Hbl) has been widely characterized as a hemolytic and cytotoxic virulence factor involved in foodborne diarrheal illness caused by Bacillus cereus. Previous studies have described the formation of Hbl complex aimed to identify toxin’s mode action. In this study, we analyzed assembly out its three individual subunits L1, L2 B soluble well putative membrane bound composition using Chinese hamster ovary (CHO) cell-free system. Subunits were either...

Incorporation of noncanonical amino acids (ncAAs) with bioorthogonal reactive groups by amber suppression allows the generation synthetic proteins desired novel properties. Such modified molecules are in high demand for basic research and therapeutic applications such as cancer treatment vivo imaging. The positioning ncAA-responsive codon within protein’s coding sequence is critical order to maintain protein function, achieve yields ncAA-containing protein, allow effective conjugation....

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 anti-CD7 RIT based on PE24 a prokaryotic eukaryotic cell-free system. The system was derived from Escherichia coli BL21 StarTM (DE3) cells transformed with plasmid encoding chaperones groEL/groES. prepared Chinese hamster ovary (CHO) that leave intact endoplasmic reticulum-derived microsomes reaction mix which...

Antibody-drug conjugates are cancer therapeutics that combine specificity and toxicity. A highly cytotoxic drug is covalently attached to an antibody directs it cells. The conjugation of the drug-linker a key point in research development as well industrial production. consensus conjugate surface-exposed part ensure maximum efficiency. However, hydrophobic nature majority drugs used antibody-drug leads increased hydrophobicity generated conjugates, resulting higher liver clearance decreased...

Abstract Cell-free protein synthesis (CFPS) enables the development of antibody conjugates, such as fluorophore conjugates and antibody-drug (ADCs), in a rapid straightforward manner. In first part, we describe cell-free antibodies containing fluorescent non-canonical amino acids (ncaa) by using pre-charged tRNA. second ncaa an orthogonal system, followed site-specific conjugation dye DyLight 650-phosphine. The expression was analyzed SDS-PAGE, autoradiography labeling in-gel fluorescence....

Abstract Difficult to express membrane proteins represent an increasing amount of therapeutic molecules. Considerable optimization is often required for downstream applications such as assay development and functional characterization. Cell-free systems emerged powerful tools the synthesis structurally functionally divergent proteins. Vesicle-based eukaryotic cell-free enable co-translational protein translocation posttranslational modifications. Hence, these provide a multitude options studies.