Production of functional antibody fragments in a vesicle-based eukaryotic cell-free translation system

Stech, Marlitt; Merk, Helmut; Schenk, Jörg A.; Stöcklein, Walter; Wüstenhagen, Doreen A.; Micheel, Burkhard; Duschl, Claus; Bier, Frank F.; Kubick, Stefan

doi:10.1016/j.jbiotec.2012.08.020

Cited by 60 publications

(68 citation statements)

References 38 publications

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“…In particular, for the synthesis of antibody fragments cell-free expression systems have gained growing attention. Recently, the production of Fab and single-chain antibody fragments (scFv) has been reported in insect cell lysates which only need moderate modification of the reaction conditions while yielding highly functional molecules [25,27]. In contrast to E. coli-based systems, these lysates do not need to be supplemented with endogenous enzymes and cofactors.…”

Section: Introductionmentioning

confidence: 99%

Cell-Free Systems: Functional Modules for Synthetic and Chemical Biology

Stech¹,

Brödel²,

Quast³

et al. 2013

Fundamentals and Application of New Bioproduction Systems

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: The main goal of cell-free protein synthesis is to produce correctly folded and functional proteins in reasonable amounts for further downstream applications. Especially for eukaryotic proteins, functionality is often directly linked to the presence of posttranslational modifications. Thus, it is of highest interest to develop novel cell-free expression systems that enable the synthesis of posttranslationally modified proteins. Here we present recent advances for the synthesis of glycoproteins, proteins containing disulfide bridges, membrane proteins, and fluorescently labeled proteins. The basis for the expression of these difficult-to-express target proteins is a translationally active cell extract which can be prepared from eukaryotic cell lines such as Spodoptera frugiperda 21 (Sf21) and Chinese hamster ovary (CHO) cells. Due to a very mild lysate preparation procedure, microsomal vesicles derived from the endoplasmic reticulum (ER) can be maintained in the eukaryotic lysate. These vesicles are translocationally active and serve as functional modules facilitating protein translocation and enrichment as well as posttranslational modification of de novo synthesized proteins. In particular, for the synthesis of membrane proteins microsomal vesicles are the essential prerequisite for the insertion of the desired protein into a biologically active membrane scaffold providing a natural environment. We anticipate that the use of such translationally active eukaryotic cell lysates containing translocationally active vesicles may solve a large number of problems still persistent when expressing eukaryotic proteins in vitro.

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Section: Introductionmentioning

confidence: 99%

Cell-Free Systems: Functional Modules for Synthetic and Chemical Biology

Stech¹,

Brödel²,

Quast³

et al. 2013

Fundamentals and Application of New Bioproduction Systems

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“…Fusion of antibody molecules to the signal sequence of honeybee melittin induced translocation and enrichment of antibody fragments in microsomal vesicles. For most scFv antibody fragments tested in these studies, signal peptide-induced translocation was shown to have a positive effect on protein functionality [10,87]. Likewise, this positive effect was also shown for the synthesis of Fab fragments by using a comparable insect cell-free translation system [90].…”

Section: Eukaryotic Cell-free Systemsmentioning

confidence: 88%

“…In 2012 and 2014, two successive studies have shown the synthesis of functional scFv antibody fragments by using the vesicle-containing insect cell-free translation system [10,87]. In comparison to other cell-free systems which have been successfully applied for the synthesis of disulfide-bonded target proteins, only moderate modifications were necessary in order to optimize the insect cell-free translation system: The abandonment of the reducing agent DTT, the addition of glutathione and the signal peptideinduced translocation of target proteins were found to be crucial factors.…”

Section: Eukaryotic Cell-free Systemsmentioning

confidence: 99%

“…In contrast to already published cell-free translation systems, neither a pretreatment of the lysate with iodoacetamide nor the addition of exogenous enzymes and chaperones were necessary in order to synthesize functional target proteins. Starting with one antibody fragment model, which binds to fluorescein [88,89], it was shown that functional scFv molecules could be synthesized based on PCR-amplified DNA templates [87]. Fusion of antibody molecules to the signal sequence of honeybee melittin induced translocation and enrichment of antibody fragments in microsomal vesicles.…”

Section: Eukaryotic Cell-free Systemsmentioning

confidence: 99%

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Cell-Free Synthesis Meets Antibody Production: A Review

Stech

Kubick

2015

Antibodies

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Engineered antibodies are key players in therapy, diagnostics and research. In addition to full size immunoglobulin gamma (IgG) molecules, smaller formats of recombinant antibodies, such as single-chain variable fragments (scFv) and antigen binding fragments (Fab), have emerged as promising alternatives since they possess different advantageous properties. Cell-based production technologies of antibodies and antibody fragments are well-established, allowing researchers to design and manufacture highly specific molecular recognition tools. However, as these technologies are accompanied by the drawbacks of being rather time-consuming and cost-intensive, efficient and powerful cell-free protein synthesis systems have been developed over the last decade as alternatives. So far, prokaryotic cell-free systems have been the focus of interest. Recently, eukaryotic in vitro translation systems have enriched the antibody production pipeline, as these systems are able to mimic the natural pathway of antibody synthesis in eukaryotic cells. This review aims to overview and summarize the advances made in the production of antibodies and antibody fragments in cell-free systems.

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“…Neben den hier beschriebenen Vorteilen bei der Synthese von Membranproteinen bieten sich die zellfreien Systeme aufgrund ihrer hervorragenden Adaptierbarkeit an die individuellen "Erfordernisse" des Zielproteins auch für die Herstellung von funktionellen Antikörperfragmenten [10,11] oder Toxinen [12,13] …”

Section: Fazitunclassified

Membranproteinsynthese: Zellfrei geht’s schneller!

et al. 2014

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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 as powerful tools for the synthesis of structurally and functionally divergent membrane proteins. Vesicle-based eukaryotic cell-free systems enable co-translational protein translocation and posttranslational modifications. Hence, these systems provide a multitude of options for membrane protein studies.

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Production of functional antibody fragments in a vesicle-based eukaryotic cell-free translation system

Cited by 60 publications

References 38 publications

Cell-Free Systems: Functional Modules for Synthetic and Chemical Biology

Cell-Free Systems: Functional Modules for Synthetic and Chemical Biology

Cell-Free Synthesis Meets Antibody Production: A Review

Membranproteinsynthese: Zellfrei geht’s schneller!

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