Rational design of a host cell protein heat precipitation step simplifies the subsequent purification of recombinant proteins from tobacco

Buyel, Johannes F.; Gruchow, Hannah M.; Boes, Alexander; Fischer, Rainer

doi:10.1016/j.bej.2014.04.015

Cited by 50 publications

(50 citation statements)

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“…Buyel and colleagues have described the benefits of processing the initial plant extract using a variety of basic treatments and additives to allow more particulates to be retained by inexpensive bag filters before expensive depth filters are challenged by the feed stream. These include a heat precipitation step to remove many soluble host cell proteins [36], the use of flocculants to aggregate smaller particulates [37,38], the use of cellulose-based filter aids to increase the retentive capacity of the filters [39] and the use of generic chromatography steps [40] based on quantitative structure-activity relationship models [41]. Statistical experimental designs were used to optimize these process options, including the order and nature of different filter types [42] and the replacement of early blade-based extraction with a commercial juicer [43].…”

Section: Downstream Processingmentioning

confidence: 99%

High-value products from plants: the challenges of process optimization

Fischer¹,

Vasil'ev²,

Twyman³

et al. 2015

Current Opinion in Biotechnology

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Section: Downstream Processingmentioning

confidence: 99%

High-value products from plants: the challenges of process optimization

Fischer¹,

Vasil'ev²,

Twyman³

et al. 2015

Current Opinion in Biotechnology

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“…Comparable values were obtained using a polytron homogenizer [35]: particle sizes ranged from 1 to 10 µm and clogged the used bag filter. We used a conventional hydropress to clarify the resulting juice and did not observe clogging.…”

Section: Discussionmentioning

confidence: 99%

“…Heat treatment has been successfully used for the purification of ELPylated hemagglutinin and ELPylated recombinant antibodies from leaves by mITC [40]. Heat treatment of plant leaves via blanching in a boiling solvent has been used to effectively remove HCPs during the purification of recombinant proteins produced in tobacco [35]. …”

Section: Discussionmentioning

confidence: 99%

Low-Tech, Pilot Scale Purification of a Recombinant Spider Silk Protein Analog from Tobacco Leaves

Heppner

Weichert

Schierhorn

et al. 2016

IJMS

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Spider dragline is used by many members of the Araneae family not only as a proteinogenic safety thread but also for web construction. Spider dragline has been shown to possess high tensile strength in combination with elastic behavior. This high tensile strength can be attributed to the presence of antiparallel β-sheets within the thread; these antiparallel β-sheets are why the protein is classified as a silk. Due to the properties of spider silk and its technical and medical uses, including its use as a suture material and as a scaffold for tissue regeneration, spider dragline is a focus of the biotechnology industry. The production of sufficient amounts of spider silk is challenging, as it is difficult to produce large quantities of fibers because of the cannibalistic behavior of spiders and their large spatial requirements. In recent years, the heterologous expression of genes coding for spider silk analogs in various hosts, including plants such as Nicotiana tabacum, has been established. We developed a simple and scalable method for the purification of a recombinant spider silk protein elastin-like peptide fusion protein (Q-/K-MaSp1-100× ELP) after heterologous production in tobacco leaves involving heat and acetone precipitation. Further purification was performed using centrifugal Inverse Transition Cycling (cITC). Up to 400 mg of highly pure spider silk protein derivatives can be isolated from six kilograms of tobacco leaves, which is the highest amount of silk protein derivatives purified from plants thus far.

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“…Although based on the principles described above for non-pharmaceutical products, the need for quality and consistency in the manufacturing process has driven researchers to identify novel approaches to remove plant-specific contaminants and to develop models to facilitate purification. For example, flocculation (Buyel and Fischer 2014a) and heat precipitation (Buyel et al 2014) have been shown to increase the efficiency of depth filtration during the purification of antibodies produced in tobacco leaves, and the filter train can also be optimized to remove plant-derived particulates more effectively (Buyel and Fischer 2014b). The behavior of the target protein and host cell proteins can be modeled to improve the overall efficiency of purification, including the use of quantitative structure-activity relationship (QSAR) models to predict how host cell proteins behave during chromatography (Buyel et al 2013b).…”

Section: Economic Barriersmentioning

confidence: 99%