High-yield production of “difficult-to-express” proteins in a continuous exchange cell-free system based on CHO cell lysates

Thoring, Lena; Dondapati, Srujan Kumar; Stech, Marlitt; Wüstenhagen, Doreen A.; Kubick, Stefan

doi:10.1038/s41598-017-12188-8

Cited by 91 publications

(97 citation statements)

References 83 publications

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“…Because the open environment enables flexibility for optimizing extract and reaction conditions and is amenable to highthroughput automation [3], cell-free gene expression (CFE) technology has found great utility in a wide range of contexts. Since their first application in deciphering the genetic code [4,5], cell-free systems have been successfully applied for the bulk production of model [6][7][8][9] and therapeutic proteins [10][11][12][13][14][15]. Beyond just protein synthesis, though, CFE technologies have evolved more generally to enable complex and diverse functions, including prototyping cellular metabolism [16][17][18] and glycosylation [19][20][21], expressing minimal synthetic cells, virus-like particles, and bacteriophages [7,[22][23][24][25][26], portable on-demand manufacturing of pharmaceuticals [27,28], incorporation of nonstandard amino acids within proteins [29][30][31][32][33], prototyping of genetic circuitry [34][35][36], and sensing viral RNAs and small molecules through rapid, low-cost, and fielddeployable molecular diagnostics [37][38][39][40][41][42]…”

Section: Introductionmentioning

confidence: 99%

Deconstructing cell-free extract preparation forin vitroactivation of transcriptional genetic circuitry

Silverman¹,

Kelley‐Loughnane

Lucks³

et al. 2018

Preprint

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Recent advances in cell-free gene expression (CFE) systems have enabled their use for a host of synthetic biology applications, particularly for rapid prototyping of genetic circuits designed as biosensors. Despite the proliferation of cell-free protein synthesis platforms, the large number of currently existing protocols for making CFE extracts muddles the collective understanding of how the method by which an extract is prepared affects its functionality. Specifically, a key goal toward developing cell-free biosensors based on native genetic regulators is activating the transcriptional machinery present in bacterial extracts for protein synthesis. However, protein yields from genes transcribed in vitro by the native Escherichia coli RNA polymerase are quite low in conventional crude extracts originally optimized for expression by the bacteriophage transcriptional machinery. Here, we show that cell-free expression of genes under bacterial σ 70 promoters is constrained by the rate of transcription in crude extracts and that processing the extract with a ribosomal run-off reaction and subsequent dialysis can alleviate this constraint. Surprisingly, these processing steps only enhance protein synthesis in genes under native regulation, indicating that the translation rate is unaffected. We further investigate the role of other common process variants on extract performance and demonstrate that bacterial transcription is inhibited by including glucose in the growth culture, but is unaffected by flash-freezing the cell pellet prior to lysis. Our final streamlined protocol for preparing extract by sonication generates extract that facilitates expression from a diverse set of sensing modalities including protein and RNA regulators. We anticipate that this work will clarify the methodology for generating CFE extracts that are active for biosensing and will encourage the further proliferation of cell-free gene expression technology for new applications.

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Section: Introductionmentioning

confidence: 99%

Deconstructing cell-free extract preparation forin vitroactivation of transcriptional genetic circuitry

Silverman¹,

Kelley‐Loughnane

Lucks³

et al. 2018

Preprint

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show abstract

“…To facilitate cap‐independent translation an IRES sequence was included upstream of the luciferase gene. As the GPR1 IRES has successfully been used in vivo in P. pastoris (Liang, Lin, Li, & Ye, ) this was chosen alongside the CrPV IRES, which shows a broad host range (Brodel et al, ; Hodgman & Jewett, ; Reavy & Moore, ; Thoring, Dondapati, Stech, Wüstenhagen, & Kubick, ). In addition, a vector with no IRES was included as a benchmark.…”

Section: Resultsmentioning

confidence: 99%

Biosensor‐assisted engineering of a high‐yield Pichia pastoris cell‐free protein synthesis platform

Polizzi

2019

Biotech & Bioengineering

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Cell‐free protein synthesis (CFPS) has recently undergone a resurgence partly due to the proliferation of synthetic biology. The variety of hosts used for cell‐free extract production has increased, which harnesses the diversity of cellular biosynthetic, protein folding, and posttranslational modification capabilities available. Here we describe a CFPS platform derived from Pichia pastoris, a popular recombinant protein expression host both in academia and the biopharmaceutical industry. A novel ribosome biosensor was developed to optimize the cell extract harvest time. Using this biosensor, we identified a potential bottleneck in ribosome content. Therefore, we undertook strain engineering to overexpress global regulators of ribosome biogenesis to increase in vitro protein production. CFPS extracts from the strain overexpressing FHL1 had a three‐fold increase in recombinant protein yield compared with those from the wild‐type X33 strain. Furthermore, our novel CFPS platform can produce complex therapeutic proteins, as exemplified by the production of human serum albumin to a final yield of 48.1 μg ml −1. Therefore, this study not only adds to the growing number of CFPS systems from diverse organisms but also provides a blueprint for rapidly engineering new strains with increased productivity in vitro that could be applied to other organisms.

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“…However, these cells have limitations when it comes to difficult-to-express proteins like overexpression of complex MPs, toxic proteins, and multi-subunit proteins as discussed above. CF systems based on CHO lysates are evolving as an alternative strategy for the expression of difficult-to-express proteins [13,[17][18][19]46]. Apart from many general advantages of CF systems, CHO-based CF systems retain most of the features of CHO cells while being more flexible due to the lack of cell membrane boundaries.…”

Section: Eukaryotic Cell-free Platformsmentioning

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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High-yield production of “difficult-to-express” proteins in a continuous exchange cell-free system based on CHO cell lysates

Cited by 91 publications

References 83 publications

Deconstructing cell-free extract preparation forin vitroactivation of transcriptional genetic circuitry

Deconstructing cell-free extract preparation forin vitroactivation of transcriptional genetic circuitry

Biosensor‐assisted engineering of a high‐yield Pichia pastoris cell‐free protein synthesis platform

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

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