Expression and secretion of the heterodimeric protein interleukin‐12 in plant cell suspension culture

Kwon, Taeho; Seo, Jae-Suk; Kim, Jae Bum; Lee, J. H.; Jang, Yong-Suk; Yang, Moon-Sik

doi:10.1002/bit.10528

“…Although secretion of large protein complexes by cultured plant cells depends on a number of factors, and it is not always feasible, several recent studies have indicated feasibility of secretion of large recombinant proteins in plant cell cultures. Notable examples include the secretion of a 155-kDa IgG1 in tobacco cell culture (albeit its modest accumulation in the medium [Sharp and Doran, 2001]) and the secretion of a 75-kDa heterodimeric protein, human interleukin-12 (hIL-12) (Kwon et al, 2003). In the event direct GFP fusion hampers protein secretion, modifications or alternatives to protein fusion may be sought.…”

Section: Discussionmentioning

confidence: 99%

High‐level secretion of functional green fluorescent protein from transgenic tobacco cell cultures: Characterization and sensing

Su

¹

,

Guan

²

,

Bugos

³

2004

Biotech & Bioengineering

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Green fluorescent protein (GFP) is useful for studying protein trafficking in plant cells. This utility could potentially be extended to develop an efficient secretory reporter system or to enable on-line monitoring of secretory recombinant protein production in plant cell cultures. Toward this end, the aim of the present study was to: (1) demonstrate and characterize high levels of secretion of fluorescent GFP from transgenic plant cell culture; and (2) examine the utility of GFP fluorescence for monitoring secreted recombinant protein production. In this study we expressed in tobacco cell cultures a secretory GFP construct made by splicing an Arabidopsis basic chitinase signal sequence to GFP. Typical extracellular GFP accumulation was 12 mg/L after 10 to 12 days of culture. The secreted GFP is functional and it accounts for up to 55% of the total GFP expressed. Findings from culture treatments with brefeldin A suggest that GFP is secreted by the cultured tobacco cells via the classical endoplasmic reticulum-Golgi pathway. Over the course of flask cultures, medium fluorescence increased with the secreted GFP concentrations that were determined using either Western blot or enzyme-linked immunoassay. Real-time monitoring of secreted GFP in plant cell cultures by on-line fluorescence detection was verified in bioreactor cultures in which the on-line culture fluorescence signals showed a linear dependency on the secreted GFP concentrations.

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“…Previous reports of bioproduction of mammalian IL-12 in plants ( GutierrezOrtega et al, 2004GutierrezOrtega et al, , 2005Kwon et al, 2003) yielded IL-12 at very low levels and demonstrated only partial IL-12 activity. In contrast, the Cramer/Dolan laboratory has developed tobacco lines that yielded high levels of murine that show equivalent bioactivity to animal cell-derived mIL-12 in both in vitro assays and in mouse vaccination studies (Liu, 2006;Liu et al, 2008).…”

mentioning

confidence: 92%

“…More than 80% of hIL-12 was secreted into the medium with less than 20% remaining in the cells (Kwon et al 2003). Although the cells continued vigorous growth, the concentration of total and secreted concentration of IL-12 dropped precipitously to about 10 mg g À1 FW, while the intracellular levels increased to about 55 mg g À1 FW at day 11, well into stationary phase.…”

mentioning

confidence: 96%

“…5C). When the main N, P, and C nutrients were measured in the culture medium, it was observed that ammonium dropped rapidly almost to zero by day 6, while Calculated from DW data in Kwon et al (2003) with a DW assumption of 10% of FW. Data taken from day 5 when IL-12 was at its highest yield.…”

mentioning

confidence: 99%

“…Gelatin (Kwon et al, 2003;Lee et al, 2002) and other protease limiting constituents added to the medium including BSA (James et al, 2000), bacitracin (Sharp and Doran, 1999), and PVP (Liu and Lee, 1999) have been proposed to act as substrates for protease thereby limiting damage to the desired secreted protein product. The addition of gelatin resulted in a fourfold increase in recovery of hIL-12 in the medium at day 5 (Kwon et al, 2003).…”

mentioning

confidence: 99%

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Production of mouse interleukin‐12 is greater in tobacco hairy roots grown in a mist reactor than in an airlift reactor

Liu

¹

,

Towler

²

,

Medrano

³

et al. 2008

Biotech & Bioengineering

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We compared the growth and productivity of a tobacco line of hairy roots that produces murine interleukin 12 (mIL-12) grown in three different culture systems: shake flasks, an airlift reactor, and a scalable mist reactor. Of the total mIL-12 produced by cultures grown in shake flasks ( approximately 434.8 microg L(-1)), almost 21% was recovered from the medium. In contrast to roots harvested from shake flasks and the mist reactor, roots were not uniformly distributed in the airlift reactor. Roots formed a dense ring around the wall of the reactor and surrounding the central rising column of fine aeration bubbles. Root quality was also better in both the shake flasks and mist reactor than in the airlift reactor. There were more pockets of dark roots in the airlift reactor suggesting some of the roots were nutrient starved. Although the best root growth (7 g DW L(-1)) was in the shake flasks, both reactors produced about the same, but less dry mass, nearly 5 g DW L(-1). Total mIL-12 concentration was highest in the mist reactor at 5.3 microg g(-1) FW, but productivity, 31 microg g(-1) FW day(-1) was highest in shake flasks. Roots grown in the mist reactor produced about 49.5% more mIL-12 than roots grown in the airlift reactor. Protease activity in the media increased steadily during culture of the roots in all three systems. The comparisons of protease activity, protein and mIL-12 levels done in the shake flask system suggest that the increase in proteases associated with progression into stationary phase is most detrimental to mIL-12 concentration. This is the first description of the design and operation of a scalable version of a mist bioreactor that uses a plastic bag. This also the first report of reasonable production levels of functional mIL-12, or any protein, produced by hairy roots grown in a mist reactor. Results will prove useful for further optimization and scale-up studies of plant-produced therapeutic proteins.

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Large‐scale Protein Production in Plants: Host Plants, Systems and Expression

Twyman

¹

2008

Protein Science Encyclopedia

1

0

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Originally published in: Molecular Farming. Edited by Rainer Fischer and Stefan Schillberg. Copyright © 2004 Wiley‐VCH Verlag GmbH & Co. KGaA Weinheim. Print ISBN: 3‐527‐30786‐9 The sections in this article are Introduction Host Species for Molecular Farming Leafy Crops Tobacco ( Nicotiana tabacum ) Tobacco ( Nicotiana benthamiana ) Alfalfa ( Medicago sativa ) White clover ( Trifolium repens ) Lettuce ( Lactuca sativa ) Spinach ( Spinacia oleracea ) Lupin ( Lupinus spp .) Dry Seed Crops Maize ( Zea mays ) Rice ( Oryza sativa ) Wheat ( Triticum aestivium ) Barley ( Hordeum vulgare ) Soybean ( Glycine max ) Pea ( Pisum sativum ) Pigeon pea ( Cajanus cajan ) Peanut ( Arachis hypogaea ) Fruit and vegetable crops Potato ( Solanum tuberosum ) Tomato ( Lycopersicon esculentum ) Carrot ( Daucus carota ) Banana ( Musa spp.) Oilcrops Rapeseed/Canola ( Brassica napus ) Falseflax ( Camelina sativa ) Safflower ( Carthamus tinctorius ) Unicellular Plants and Aquatic Plants Maintained in Bioreactors Chlamydomonas reinhardtii Physcomitrella patens Duckweed ( Lemna minor ) Non‐cultivated Model Plants Arabidopsis thaliana Expression systems for molecular farming Transgenic plants Transplastomic plants Virus‐infected plants Transiently transformed leaves Hydroponic cultures Hairy roots Shooty teratomas Suspension cell cultures Expression strategies and protein yields Conclusions

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Expression and secretion of the heterodimeric protein interleukin‐12 in plant cell suspension culture

Cited by 60 publications

References 31 publications

High‐level secretion of functional green fluorescent protein from transgenic tobacco cell cultures: Characterization and sensing

High‐level secretion of functional green fluorescent protein from transgenic tobacco cell cultures: Characterization and sensing

Production of mouse interleukin‐12 is greater in tobacco hairy roots grown in a mist reactor than in an airlift reactor

Large‐scale Protein Production in Plants: Host Plants, Systems and Expression

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