High-throughput screening of biomolecules using cell-free gene expression systems

Contreras-Llano, Luis E; Tan, Cheemeng

doi:10.1093/synbio/ysy012

Cited by 58 publications

(44 citation statements)

References 124 publications

(104 reference statements)

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“…The parallel synthesis of several proteins directly onto an immobilizing surface can circumvent these drawbacks. Different methods are described, such as PISA (Protein In Situ Array), NAPPA (Nucleic Acid Programmable Protein Array), and DAPA (DNA to Protein Array) [91]. All approaches have in common that the transcription, translation, and immobilization take place simultaneously in situ (on-chip).…”

Section: Applications Of Cell-free Protein Synthesismentioning

confidence: 99%

Application of Cell-Free Protein Synthesis for Faster Biocatalyst Development

2019

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Cell-free protein synthesis (CFPS) has become an established tool for rapid protein synthesis in order to accelerate the discovery of new enzymes and the development of proteins with improved characteristics. Over the past years, progress in CFPS system preparation has been made towards simplification, and many applications have been developed with regard to tailor-made solutions for specific purposes. In this review, various preparation methods of CFPS systems are compared and the significance of individual supplements is assessed. The recent applications of CFPS are summarized and the potential for biocatalyst development discussed. One of the central features is the high-throughput synthesis of protein variants, which enables sophisticated approaches for rapid prototyping of enzymes. These applications demonstrate the contribution of CFPS to enhance enzyme functionalities and the complementation to in vivo protein synthesis. However, there are different issues to be addressed, such as the low predictability of CFPS performance and transferability to in vivo protein synthesis. Nevertheless, the usage of CFPS for high-throughput enzyme screening has been proven to be an efficient method to discover novel biocatalysts and improved enzyme variants.

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Section: Applications Of Cell-free Protein Synthesismentioning

confidence: 99%

Application of Cell-Free Protein Synthesis for Faster Biocatalyst Development

2019

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“…Successful synthesis of different antibody formats, including single-chain variable fragments (scFvs), Fab fragments, as well as complete IgGs, has already been shown in E. coli [114,115], Sf21 [10,116], reticulocyte [110], wheat germ [117], and CHO CF systems [46,75,118]. Furthermore, the upscaling of CF reactions to the liter-scale [25,115] as well as downscaling [119] and high-throughput applications [120] have been demonstrated.…”

Section: Antibody Productionmentioning

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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“…It has been reported, that 1 mL of such an emulsion can hold as many as 10 10 droplets [170]. Adaptations of this technique (e.g., STABLE [172], SNAP [173]) have been summarized in a recent review [174]. Although IVC is a technology especially handy for in vitro enzyme evolution [175], it has also been applied for screening peptide libraries [176,177].…”

Section: Acellular Approachmentioning

confidence: 99%

Evolving a Peptide: Library Platforms and Diversification Strategies

Bozovičar

Bratkovič

2019

IJMS

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Peptides are widely used in pharmaceutical industry as active pharmaceutical ingredients, versatile tools in drug discovery, and for drug delivery. They find themselves at the crossroads of small molecules and proteins, possessing favorable tissue penetration and the capability to engage into specific and high-affinity interactions with endogenous receptors. One of the commonly employed approaches in peptide discovery and design is to screen combinatorial libraries, comprising a myriad of peptide variants of either chemical or biological origin. In this review, we focus mainly on recombinant peptide libraries, discussing different platforms for their display or expression, and various diversification strategies for library design. We take a look at well-established technologies as well as new developments and future directions.

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High-throughput screening of biomolecules using cell-free gene expression systems

Cited by 58 publications

References 124 publications

Application of Cell-Free Protein Synthesis for Faster Biocatalyst Development

Application of Cell-Free Protein Synthesis for Faster Biocatalyst Development

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

Evolving a Peptide: Library Platforms and Diversification Strategies

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