Cell-Free Protein Synthesis Systems Derived from Cultured Mammalian Cells

Brödel, Andreas K.; Wüstenhagen, Doreen A.; Kubick, Stefan

doi:10.1007/978-1-4939-2230-7_7

Cited by 30 publications

(29 citation statements)

References 24 publications

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“…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. However, the reports have been confined to autoradiography studies.…”

Section: Discussionmentioning

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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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%

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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“…The translation of hBMP2 is initiated by a 5 -end cap region and that of the EGFP by an IRES sequence. In addition, a linear template for cell-free expression was generated by a two-step PCR procedure, as previously described [29]. pMK-CRPV-Mel-hBMP2 (3.9 kb, Fig.…”

Section: Dna Constructsmentioning

confidence: 99%

“…CHO cell lysate preparation and cell-free protein synthesis were performed as previously described [26,29]. Briefly, cells were suspended in lysis buffer (40 mM HEPES, pH 7.5, 100 mM NaOAc, 4 mM DTT) at approximately 5 × 10 8 cells/mL and syringed through a 20-gauge needle.…”

Section: Cell-free Protein Synthesismentioning

confidence: 99%

“…Plasmid concentration and quality (> 80 % supercoiled topology) were determined by A 260/280 ratio (> 1.8) and agarose gel electrophoresis. In addition, a linear template for cell-free expression was generated by a two-step PCR procedure, as previously described [29]. A schematic overview of the procedure, including the introduction of the necessary regulatory sequences for cell-free protein synthesis in CHO cell lysates, is given in Fig.…”

Section: Dna Constructsmentioning

confidence: 99%

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Comparison of cell‐based versus cell‐free mammalian systems for the production of a recombinant human bone morphogenic growth factor

Jérôme

Thoring

Salzig

et al. 2017

Engineering in Life Sciences

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The human bone morphogenetic protein-2 (hBMP2) is a glycoprotein, which induces de novo bone formation. Here, recombinant production in stably transfected Chinese Hamster Ovary (CHO) cells is compared to transient expression in Human Embryo Kidney (HEK) cells and cell-free synthesis in CHO cell lysates containing microsomal structures as sites of post-translational processing. In case of the stably transfected cells, growth rates and viabilities were similar to those of the parent cells, while entry into the death phase of the culture was delayed. The maximum achievable rhBMP2 concentration in these cultures was 153 pg/mL. Up to 280 ng/mL could be produced in the transient expression system. In both cases the rhBMP-2 was found to interact with the producer cells, which presumably contributed to the low yields. In the cell-free system, hBMP2 yields could be increased to almost 40 μg/mL, reached within three hours. The cell-free system thus approached productivities for the active (renatured) protein previously only recorded for bacterial hosts, while assuring comprehensive post-translational processing

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

Tran

Gurramkonda

Cooper

et al. 2017

Biotech & Bioengineering

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The use of cell-free systems to produce recombinant proteins has grown rapidly over the past decade. In particular, cell-free protein synthesis (CFPS) systems based on mammalian cells provide alternative methods for the production of many proteins, including those that contain disulfide bonds, glycosylation, and complex structures such as monoclonal antibodies. In the present study, we show robust production of turbo green fluorescent protein (tGFP) and streptokinase in a cell-free system using instrumented mini-bioreactors for highly reproducible protein production. We achieved recombinant protein production (∼600 μg/ml of tGFP and 500 μg/ml streptokinase) in 2.5 hr of expression time, comparable to previously reported yields for cell-free protein expression. Also, we demonstrate the use of two different affinity tags for product capture and compare those to a tag-free self-cleaving intein capture technology. The intein purification method provided a product recovery of 86%, compared with 52% for conventionally tagged proteins, while resulting in a 30% increase in total units of activity of purified recombinant streptokinase compared with conventionally tagged proteins. These promising beneficial features combined with the intein technology makes feasible the development of dose-level production of therapeutic proteins at the point-of-care. K E Y W O R D Scell-free in vitro translation, green fluorescent protein, intein technology, mini-bioreactors

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Cell-Free Protein Synthesis Systems Derived from Cultured Mammalian Cells

Cited by 30 publications

References 24 publications

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

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

Comparison of cell‐based versus cell‐free mammalian systems for the production of a recombinant human bone morphogenic growth factor

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

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