Tobacco BY-2 Media Component Optimization for a Cost-Efficient Recombinant Protein Production

Häkkinen, Suvi T.; Reuter, Lauri; Nuorti, Ninni; Joensuu, Jussi; Rischer, Heiko; Ritala, Anneli

doi:10.3389/fpls.2018.00045

Cited by 39 publications

(29 citation statements)

References 29 publications

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“…Noticeably, massive fluorescent aggregates of irregular shapes measuring 5–15 μm in length occurred in the BFA‐treated root cells (Figure c–f). Some of these aggregates, particularly those around the nuclei, were like the perinuclear pleiomorphic aggregates as described earlier in the BFA‐treated BY‐2 cells (Ritzenthaler et al ., ), whereas the others were likely protein bodies formed due to concentrating of the EGFP products in ER, as reported recently for some GFP‐derived proteins overexpressed in tobacco leaves and BY‐2 cells (Hakkinen et al ., ; Saberianfar et al ., ). These changes indicated that BFA treatment successfully blocked the transport of (SP4) 18 ‐EGFP and (SP) 32 ‐EGFP transgene products from ER to Golgi apparatus.…”

Section: Resultsmentioning

confidence: 99%

Engineering ‘designer’ glycomodules for boosting recombinant protein secretion in tobacco hairy root culture and studying hydroxyproline‐O‐glycosylation process in plants

Wright

Wang

Karki

et al. 2018

Plant Biotechnology Journal

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Summary The key technical bottleneck for exploiting plant hairy root cultures as a robust bioproduction platform for therapeutic proteins has been low protein productivity, particularly low secreted protein yields. To address this, we engineered novel hydroxyproline (Hyp)‐ O ‐glycosylated peptides (Hyp GP s) into tobacco hairy roots to boost the extracellular secretion of fused proteins and to elucidate Hyp‐ O ‐glycosylation process of plant cell wall Hyp‐rich glycoproteins. Hyp GP s representing two major types of cell wall glycoproteins were examined: an extensin module consisting of 18 tandem repeats of ‘Ser‐Hyp‐Hyp‐Hyp‐Hyp’ motif or ( SP 4) 18 and an arabinogalactan protein module consisting of 32 tandem repeats of ‘Ser‐Hyp’ motif or ( SP ) 32 . Each module was expressed in tobacco hairy roots as a fusion to the enhanced green fluorescence protein ( EGFP ). Hairy root cultures engineered with a Hyp GP module secreted up to 56‐fold greater levels of EGFP , compared with an EGFP control lacking any Hyp GP module, supporting the function of Hyp GP modules as a molecular carrier in promoting efficient transport of fused proteins into the culture media. The engineered ( SP 4) 18 and ( SP ) 32 modules underwent Hyp‐ O ‐glycosylation with arabino‐oligosaccharides and arabinogalactan polysaccharides, respectively, which were essential in facilitating secretion of the fused EGFP protein. Distinct non‐Hyp‐ O ‐glycosylated ( SP 4) 18 ‐ EGFP and ( SP ) 32 ‐ EGFP intermediates were consistently accumulated within the root tissues, indicating a rate‐limiting trafficking and/or glycosylation of the engineered Hyp GP modules. An updated model depicting the intracellular trafficking, Hyp‐ O ‐glycosylation and extracellular secretion of extensin‐styled ( SP 4) 18 module and AGP ‐styled ( SP ) 32 module is proposed.

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Section: Resultsmentioning

confidence: 99%

Engineering ‘designer’ glycomodules for boosting recombinant protein secretion in tobacco hairy root culture and studying hydroxyproline‐O‐glycosylation process in plants

Wright

Wang

Karki

et al. 2018

Plant Biotechnology Journal

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“…Total soluble protein (TSP) was extracted from 100 to 200 mg fresh mass of the infused PCPs and Agrobacterium cells harbouring the respective expression vectors. The cells were frozen in liquid nitrogen in microfuge tubes, ground by bead beating using FastPrep-24 Instrument (MP Biomedicals), dissolved in a double volume of extraction buffer (1 × phosphate-buffered saline; 137 mM NaCl, 2.7 mM KCl, 10 mM Na 2 HPO 4 , 1.8 mM KH 2 PO 4 , pH 7.4), 1 mM EDTA (Sigma Aldrich, St. Louis, MO, United States), 1 × protease inhibitor cocktail (ThermoFisher Scientific) (Häkkinen et al 2018), and again subjected to homogenization using the FastPrep-24 Instrument. The extracts were centrifuged (10,000g at 4 °C) for 15 min and used for further analyses.…”

Section: Protein Extraction and Quantificationmentioning

confidence: 99%

“…takes place under fully controlled sterile conditions in chemically defined media (Häkkinen et al 2018), which allows the production of recombinant proteins according to the current good manufacturing practice (Fischer et al 2005). The high-value product can also be more easily recovered and purified, especially when the product is secreted into the culture medium (Fischer et al 1999).…”

Section: Introductionmentioning

confidence: 99%

Transient protein expression in tobacco BY-2 plant cell packs using single and multi-cassette replicating vectors

et al. 2020

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Key message This is the first evidence that replicating vectors can be successfully used for transient protein expression in BY-2 plant cell packs. Abstract Transient recombinant protein expression in plants and recently also plant cell cultures are of increasing interest due to the speed, safety and scalability of the process. Currently, studies are focussing on the design of plant virus-derived vectors to achieve higher amounts of transiently expressed proteins in these systems. Here we designed and tested replicating single and multi-cassette vectors that combine elements for enhanced replication and hypertranslation, and assessed their ability to express and particularly co-express proteins by Agrobacterium-mediated transient expression in tobacco BY-2 plant cell packs. Substantial yields of green and red fluorescent proteins of up to ~ 700 ng/g fresh mass were detected in the plant cells along with position-dependent expression. This is the first evidence of the ability of replicating vectors to transiently express proteins in BY-2 plant cell packs.

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“…We postulate that RLK1ox cells may compete in producing proteins more than wildtype cells based on such characteristics as smaller cell size, fast dividing characteristic, and powerful proliferation because it is well known that BY-2 cells can be used as host cells for the large-scale production of recombinant proteins [45].…”

Section: Discussionmentioning

confidence: 99%

Decrease in cell size and cell wall components under ectopic expression of the CaRLK1 gene

Lee¹,

Kim

Jeong

et al. 2020

Preprint

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Abstract BackgroundThe Capsicum annuum receptor-like kinase 1 (CaRLK1) gene encodes a transmembrane protein with a cytoplasmic kinase domain and an extracellular domain. It functions as a negative regulator of plant cell death. Ectopic expression of CaRLK1 showed the hypoxia-resistance and enhanced cell division and proliferation. In this study, it was investigated which genes were controlled by ectopic expression of CaRLK1 because it decreased its average cell size of transgenic RLK1ox cells compared with that of wild-type cells (BY-2). ResultsThe average diameter (AD) of the protoplast of RLK1ox cells was about 10 µm shorter than the AD of BY-2 cells. The diameter distribution of the circular protoplasts is mainly from 20 to 40 μm (71.7%) in RLK1ox cells, whereas from 30 to 50 μm (73.0%) in BY-2 cells. Furthermore, cell volume of RLK1ox cells is also 2.5-times smaller than that of BY-2 cells. Smaller cell size of the RLK1ox cells may be related with the inhibited cell expansion because expressions of 12 expansin A genes, 6 expansin-like B genes, 2 ACC synthase genes, and 5 ACC oxidase genes were suppressed, but expressions of 7 gibberellin 2-oxidase genes were induced. The cell walls between two RLK1ox cells are approximately 138.83±4.12 nm thick on average, while those between two BY-2 cells are approximately 156.58±4.54 nm thick. Furthermore, the total content of neutral sugars in the cell wall of RLK1ox cells is less than that of BY-2 cells (about 25%). The RLK1ox cells contained 30% less glucose content than did the BY-2 cells. Thinner cell wall of the RLK1ox cells is related with the decreased cellulose biosynthesis and hemicellulose biosynthesis. Expressions of 7 CESA genes, 5 sucrose synthase 1 genes, 3 mannose-1-phosphate guanyltransferase genes, and 6 glucomannan-4-beta-mannosyltransferase genes were suppressed. The suppressed expressions of 14 polygalacturonase genes may also contribute to make the cell wall of RLK1ox cell thinner than that of BY-2 cell. ConclusionOf special emphasis is its impact of CaRLK1 gene on cell size control and cell wall thickness. Smaller cell size of the RLK1ox cells correlates with the inhibited cell expansion.

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Tobacco BY-2 Media Component Optimization for a Cost-Efficient Recombinant Protein Production

Cited by 39 publications

References 29 publications

Engineering ‘designer’ glycomodules for boosting recombinant protein secretion in tobacco hairy root culture and studying hydroxyproline‐O‐glycosylation process in plants

Engineering ‘designer’ glycomodules for boosting recombinant protein secretion in tobacco hairy root culture and studying hydroxyproline‐O‐glycosylation process in plants

Transient protein expression in tobacco BY-2 plant cell packs using single and multi-cassette replicating vectors

Decrease in cell size and cell wall components under ectopic expression of the CaRLK1 gene

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