Cell‐free production of a therapeutic protein: Expression, purification, and characterization of recombinant streptokinase using a CHO lysate

Tran, Kevin; Gurramkonda, Chandrasekhar; Cooper, Merideth A.; Pilli, Manohar; Taris, Joseph E.; Selock, Nicholas; Han, Tzu-Chiang; Tolosa, Michael; Zuber, Adil; Peñalber-Johnstone, Chariz; Dinkins, Christina; Pezeshk, Niloufar; Kostov, Yordan; Frey, Douglas D.; Tolosa, Leah; Wood, David; Rao, Govind

doi:10.1002/bit.26439

Cited by 39 publications

(26 citation statements)

References 29 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…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

View full text Add to dashboard Cite

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.

show abstract

Section: Introductionmentioning

confidence: 99%

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

Silverman¹,

Kelley‐Loughnane

Lucks³

et al. 2018

Preprint

View full text Add to dashboard Cite

show abstract

“…The batch method has been applied for Escherichia coli CFPS, where the cell-free reaction was carried out in a simple Eppendorf tube. Moreover, a recent demonstration of recombinant streptokinase production using cell-free system yielded 500 μg/ml in a controlled bioreactor (Tran et al, 2018). 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.…”

mentioning

confidence: 99%

“…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%

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

Gurramkonda

Rao

Borhani

et al. 2018

Biotech & Bioengineering

Self Cite

View full text Add to dashboard Cite

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.

show abstract

“…The procedure for preparing the reaction mix was adopted from previously published work(Peñalber-Johnstone et al, 2017;Tran et al, 2018) and slightly modified as follows: a 1 ml vial of IVT CHO lysate was thawed and reconstituted with 435 µl nuclease free water, 5 µl of GADD34myc, 400 µl of reaction mix (with DTT) and finally 160 µl (containing 80 µg) solution The IVT system uses a 10 kDa MWCO Slide-A-Lyzer cassette with 0.5-3 ml capacity as a modified bioreactor device which provides a constant supply of energy regenerating substrates to maintain the reaction while removing toxic by-products.…”

mentioning

confidence: 99%

“…The IVT system uses a 10 kDa MWCO Slide-A-Lyzer cassette with 0.5-3 ml capacity as a modified bioreactor device which provides a constant supply of energy regenerating substrates to maintain the reaction while removing toxic by-products. The procedure for preparing the reaction mix was adopted from previously published work(Peñalber-Johnstone et al, 2017;Tran et al, 2018) and slightly modified as follows: a 1 ml vial of IVT CHO lysate was thawed and reconstituted with 435 µl nuclease free water, 5 µl of GADD34myc, 400 µl of reaction mix (with DTT) and finally 160 µl (containing 80 µg) solution…”

mentioning

confidence: 99%

Low‐cost customizable microscale toolkit for rapid screening and purification of therapeutic proteins

Andar

Deldari

Gutierrez

et al. 2018

Biotech & Bioengineering

Self Cite

View full text Add to dashboard Cite

Biopharmaceutical separations require tremendous amounts of optimization to achieve acceptable product purity. Typically, large volumes of reagents and biological materials are needed for testing different parameters, thus adding to the expense of biopharmaceutical process development. This study demonstrates a versatile and customizable microscale column (µCol) for biopharmaceutical separations using immobilized metal affinity chromatography (IMAC) as an example application to identify key parameters. µCols have excellent precision, efficiency, and reproducibility, can accommodate any affinity, ion-exchange or size-exclusion-based resin and are compatible with any high-performance liquid chromatography (HPLC) system. µCols reduce reagent amounts, provide comparable purification performance and high-throughput, and are easy to automate compared with current conventional resin columns. We provide a detailed description of the fabrication methods, resin packing methods, and µCol validation experiments using a conventional HPLC system. Finite element modeling using COMSOL Multiphysics was used to validate the experimental performance of the µCols. In this study, µCols were used for improving the purification achieved for granulocyte colony stimulating factor (G-CSF) expressed using a cell-free CHO in vitro translation (IVT) system and were compared to a conventional 1 ml IMAC column. Experimental data revealed comparable purity with a 10-fold reduction in the amount of buffer, resin, and purification time for the μCols compared with conventional columns for similar protein yields. K E Y W O R D Schromatography, granulocyte colony stimulating factor, immobilized metal affinity chromatography, microfluidics, protein purification

show abstract

Cell‐free production of a therapeutic protein: Expression, purification, and characterization of recombinant streptokinase using a CHO lysate

Cited by 39 publications

References 29 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

Low‐cost customizable microscale toolkit for rapid screening and purification of therapeutic proteins

Contact Info

Product

Resources

About