Cell-free synthesis of functional antibodies using a coupled in vitro transcription-translation system based on CHO cell lysates

Stech, Marlitt; Николаева, О.Е.; Thoring, Lena; Stöcklein, Walter; Wüstenhagen, Doreen A.; Hust, Michael; Dübel, Stefan; Kubick, Stefan

doi:10.1038/s41598-017-12364-w

Cited by 57 publications

(63 citation statements)

References 60 publications

(62 reference statements)

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“…Most progress has occurred in CFE systems generated from Escherichia coli strains engineered for protein production, largely due to the bacterium's well-characterized genetics and metabolism [1]. However, there has been recent progress in adapting CFE protocols to make lysates from eukaryotic and non-model organisms, including yeast [43,44], Gram-positive bacteria [45,46], plants [47,48], and mammalian cells [49][50][51]. CFE technology is therefore now at the point of expanding beyond specialist laboratories and becoming a major toolbox throughout synthetic biology research, application, and recently, education [34,52,53].…”

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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“…In cell-free systems, core glycosylation can be achieved by supplementing extracts with microsomal fractions (Walter & Blobel, 1983). An insect cell-free system offers core protein glycosylation without the need for supplementing the microsomes in the reaction (Stech et al, 2014;Tarui et al, 2001). Recent studies reported the expression of recombinant erythropoietin using microsomes in CHO cell-free system showing the presence of glycosylated and unglycosylated forms (Brodel et al, 2013(Brodel et al, , 2014(Brodel et al, , 2015.…”

Section: Discussionmentioning

confidence: 99%

“…Investigators (Table 1) have used the E. coli CFPS, insect CFPS and CHO CFPS systems for the expression of human EPO (Ahn, Choi, & Kim, 2005;Ahn, Hwang, Lee, Choi, & Kim, 2007;Brodel, Sonnabend, & Kubick, 2014;Brodel, Wustenhagen, & Kubick, 2015;Stech et al, 2014). However, no further optimization studies were reported.…”

mentioning

confidence: 99%

“…In the dialysis method, which is also known as the continuous nutrient exchange system, the cell-free transcription and translation reactions are performed in a small reaction chamber that is separated by a dialysis membrane. Using the optimized dialysis system, expression of a monoclonal antibody and other immunoglobins using CHO CFPS, and the expression of tissue plasminogen activator (tPA) using insect CFPS (Martin et al, 2017;Stech et al, 2014Stech et al, , 2017. Moreover, a recent demonstration of recombinant streptokinase production using cell-free system yielded 500 μg/ml in a controlled bioreactor (Tran et al, 2018).…”

mentioning

confidence: 99%

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Improving the recombinant human erythropoietin glycosylation using microsome supplementation in CHO cell‐free system

Gurramkonda

Rao

Borhani

et al. 2018

Biotech & Bioengineering

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Cell-Free Protein Synthesis (CFPS) offers many advantages for the production of recombinant therapeutic proteins using the CHO cell-free system. However, many complex proteins are still difficult to express using this method. To investigate the current bottlenecks in cell-free glycoprotein production, we chose erythropoietin (40% glycosylated), an essential endogenous hormone which stimulates the development of red blood cells. Here, we report the production of recombinant erythropoietin (EPO) using CHO cell-free system. Using this method, EPO was expressed and purified with a twofold increase in yield when the cell-free reaction was supplemented with CHO microsomes. The protein was purified to near homogeneity using an ion-metal affinity column. We were able to analyze the expressed and purified products (glycosylated cell-free EPO runs at 25-28 kDa, and unglycosylated protein runs at 20 kDa on an SDS-PAGE), identifying the presence of glycan moieties by PNGase shift assay. The purified protein was predicted to have ∼2,300 IU in vitro activity. Additionally, we tested the presence and absence of sugars on the cell-free EPO using a lectin-based assay system. The results obtained in this study indicate that microsomes augmented in vitro production of the glycoprotein is useful for the rapid production of single doses of a therapeutic glycoprotein drug and to rapidly screen glycoprotein constructs in the development of these types of drugs. CFPS is useful for implementing a lectin-based method for rapid screening and detection of glycan moieties, which is a critical quality attribute in the industrial production of therapeutic glycoproteins.

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“…The addition of anti-spliced leader oligonucleotide to L. tarentolae cell extracts suppressed the translation of endogenous L. tarentolae mRNAs, thus increasing the translation efficiency of exogenously supplied mRNA [65]. Using the ER-specific signal sequence of honeybee melittin (melittin signal peptide) instead of the native signal peptide increased the translocation of synthesized proteins such as WNT proteins, single-chain antibody variable fragments, and the hTLR9-ectodomain into microsomes in the case of Sf21 and CHO-based CF systems [18,59,74,75].…”

Section: Gene Designmentioning

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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Cell-free synthesis of functional antibodies using a coupled in vitro transcription-translation system based on CHO cell lysates

Cited by 57 publications

References 60 publications

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

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

Improving the recombinant human erythropoietin glycosylation using microsome supplementation in CHO cell‐free system

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

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