Cell‐free production of pore forming toxins: Functional analysis of thermostable direct hemolysin from <i>Vibrio parahaemolyticus</i>

Dondapati, Srujan Kumar; Wüstenhagen, Doreen A.; Strauch, Eckhard; Kubick, Stefan

doi:10.1002/elsc.201600259

Cited by 6 publications

(3 citation statements)

References 32 publications

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“…CF systems derived from eukaryotic lysates equipped with endogenous microsomes (e.g., Sf21, CHO, cultured human cells, and Tobacco-BY2) satisfy all the necessary requirements for proper folding of MPs. The microsomes offer a native environment and intact translocon machinery for a proper embedment and folding of MPs [13,18,19,45,46,51,59]. There are continuing efforts in analyzing the functionality of microsomal reconstituted MPs, indicated by a good number of publications reporting on this reconstitution strategy, which should help the pharmaceutical industry to develop more dynamic drug screening assays involving MPs [9,46,83].…”

Section: Cell-free Systems For the Synthesis Of Membrane Proteinsmentioning

confidence: 99%

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

et al. 2020

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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. Cell-free systems are emerging as an attractive alternative for the production of proteins due to their flexible nature without any cell membrane constraints. In a bioproduction context, open systems based on cell lysates derived from different sources, and with batch-to-batch consistency, have acted as a catalyst for cell-free synthesis of target proteins. Most importantly, proteins can be processed for downstream applications like purification and functional analysis without the necessity of transfection, selection, and expansion of clones. In the last 5 years, there has been an increased availability of new cell-free lysates derived from multiple organisms, and their use for the synthesis of a diverse range of proteins. Despite this progress, major challenges still exist in terms of scalability, cost effectiveness, protein folding, and functionality. In this review, we present an overview of different cell-free systems derived from diverse sources and their application in the production of a wide spectrum of proteins. Further, this article discusses some recent progress in cell-free systems derived from Chinese hamster ovary and Sf21 lysates containing endogenous translocationally active microsomes for the synthesis of membrane proteins. We particularly highlight the usage of internal ribosomal entry site sequences for more efficient protein production, and also the significance of site-specific incorporation of non-canonical amino acids for labeling applications and creation of antibody drug conjugates using cell-free systems. We also discuss strategies to overcome the major challenges involved in commercializing cell-free platforms from a laboratory level for future drug development. Key PointsCell-free protein synthesis has the potential to become an alternative method for the rapid and highly parallel expression of a diverse range of proteins.Cell-free systems utilize the translation machinery of the cells, bypassing the constraints of the cell membrane and thus offer openness and configurational flexibility even for the synthesis of difficult-to-express proteins.In comparison to traditional approaches, cell-free systems can be time-saving, from initial synthesis to downstream applications.

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Section: Cell-free Systems For the Synthesis Of Membrane Proteinsmentioning

confidence: 99%

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

et al. 2020

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“…Chang et al, 2015; Gao et al, 2019). Highly toxic molecules that would otherwise kill living cells can also be produced with in vitro transcription/translation (IVTT) systems encapsulated in SCs (Dondapati et al, 2018; Orth et al, 2011; Salehi et al, 2016). Composing SCs of other exotic chemistry can therefore endow them with unique advantages in terms of stability as well as the functions they can enact inside the body.…”

Section: Introductionmentioning

confidence: 99%

Synthetic cells in biomedical applications

Sato

Zajkowski

Moser³

et al. 2021

WIREs Nanomed Nanobiotechnol

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Synthetic cells are engineered vesicles that can mimic one or more salient features of life. These features include directed localization, sense‐and‐respond behavior, gene expression, metabolism, and high stability. In nanomedicine, many of these features are desirable capabilities of drug delivery vehicles but are difficult to engineer. In this focus article, we discuss where synthetic cells offer unique advantages over nanoparticle and living cell therapies. We review progress in the engineering of the above life‐like behaviors and how they are deployed in nanomedicine. Finally, we assess key challenges synthetic cells face before being deployed as drugs and suggest ways to overcome these challenges. This article is categorized under: Therapeutic Approaches and Drug Discovery > Emerging Technologies Biology‐Inspired Nanomaterials > Lipid‐Based Structures

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“…Cell-free protein synthesis (CFPS) is an alternative as proteins synthesized in a eukaryotic system can be directly used for functional assessments without the need for purification. CFPS is a time-and cost-efficient way to produce single proteins, glycoproteins, protein-complexes as well as "difficult-to express" proteins such as membrane proteins and toxic proteins in a well-defined model system [14][15][16][17][18][19][20][21]. Single proteins can be synthesized and functionally characterized individually as well as in a mixture of different proteins.…”

Section: Introductionmentioning

confidence: 99%

The Pore-Forming Hemolysin BL Enterotoxin from Bacillus cereus: Subunit Interactions in Cell-Free Systems

Ramm

Stech

Zemella

et al. 2021

Toxins

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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 the Hbl complex and aimed to identify the toxin’s mode of action. In this study, we analyzed the assembly of Hbl out of its three individual subunits L1, L2 and B in a soluble as well as a putative membrane bound composition using a Chinese hamster ovary (CHO) cell-free system. Subunits were either coexpressed or synthesized individually in separate cell-free reactions and mixed together afterwards. Hemolytic activity of cell-free synthesized subunits was demonstrated on 5% sheep blood agar and identified both synthesis procedures, coexpression as well as individual synthesis of each subunit, as functional for the synthesis of an active Hbl complex. Hbl’s ability to perforate cell membranes was evaluated using a propidium iodide uptake assay. These data suggested that coexpressed Hbl subunits augmented cytotoxic activity with increasing concentrations. Further, a pre-pore-complex of L1-L2 showed cytotoxic effects suggesting the possibility of an interaction between the cell membrane and the pre-pore-complex. Overall, this study shows that cell-free protein synthesis is a fast and efficient way to study the assembly of multiple protein subunits in soluble as well as vesicular fractions.

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

Cited by 6 publications

References 32 publications

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

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

Synthetic cells in biomedical applications

The Pore-Forming Hemolysin BL Enterotoxin from Bacillus cereus: Subunit Interactions in Cell-Free Systems

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