Tomato is a suitable material for producing recombinant miraculin protein in genetically stable manner

Yano, Masahiro; Hirai, Tadayoshi; Kato, Kazuhisa; Hiwasa-Tanase, Kyoko; Fukuda, Naoya; Ezura, Hiroshi

doi:10.1016/j.plantsci.2010.02.016

Cited by 37 publications

(28 citation statements)

References 19 publications

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“…The cost of downstream processing of plant‐derived recombinant proteins is reported to account for about 80% of the total expenditure (Buonaguro and Butler‐Ransohoff, ; Kusnadi et al ., ). However, using watery tissues such as tomatoes as the production platforms has the potential of reducing the cost, as they are easier to extract than dry tissues such as cereals (Schillberg et al ., ; Yano et al ., ). The present study shows that purifying r‐protein from sugarcane juice not only provides appreciable yield but can also ultimately reduce the purification cost.…”

Section: Discussionmentioning

confidence: 97%

Vacuolar targeting of r‐proteins in sugarcane leads to higher levels of purifiable commercially equivalent recombinant proteins in cane juice

Palaniswamy

Syamaladevi

Mohan

et al. 2015

Plant Biotechnology Journal

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SummarySugarcane is an ideal candidate for biofarming applications because of its large biomass, rapid growth rate, efficient carbon fixation pathway and a well-developed storage tissue system. Vacuoles occupy a large proportion of the storage parenchyma cells in the sugarcane stem, and the stored products can be harvested as juice by crushing the cane. Hence, for the production of any high-value protein, it could be targeted to the lytic vacuoles so as to extract and purify the protein of interest from the juice. There is no consensus vacuolar-targeting sequence so far to target any heterologous proteins to sugarcane vacuole. Hence, in this study, we identified an Nterminal 78-bp-long putative vacuolar-targeting sequence from the N-terminal domain of unknown function (DUF) in Triticum aestivum 6-SFT (sucrose: fructan 6-fructosyl transferase). In this study, we have generated sugarcane transgenics with gene coding for the green fluorescent protein (GFP) fused with the vacuolar-targeting determinants at the N-terminal driven by a strong constitutive promoter (Port ubi882) and demonstrated the targeting of GFP to the vacuoles. In addition, we have also generated transgenics with His-tagged b-glucuronidase (GUS) and aprotinin targeted to the lytic vacuole, and these two proteins were isolated and purified from the transgenic sugarcane and compared with commercially available protein samples. Our studies have demonstrated that the novel vacuolar-targeting determinant could localize recombinant proteins (r-proteins) to the vacuole in high concentrations and such targeted r-proteins can be purified from the juice with a few simple steps.

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Section: Discussionmentioning

confidence: 97%

Vacuolar targeting of r‐proteins in sugarcane leads to higher levels of purifiable commercially equivalent recombinant proteins in cane juice

Palaniswamy

Syamaladevi

Mohan

et al. 2015

Plant Biotechnology Journal

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“…Among the miraculinaccumulating transgenic tomato lines, a line that we named 56B had a single copy of the miraculin transgene and accumulated miraculin to high levels in the T 0 generation. Line 56B was propagated up to the T 5 generation, which corresponds to six generations (Yano et al 2010). That study showed that miraculin transgene inheritance and expression were stable through multiple generations in transgenic tomatoes.…”

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confidence: 99%

Gene dosage and genetic background affect miraculin accumulation in transgenic tomato fruits

Kim

Hirai

Kato

et al. 2010

Plant Biotechnology

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Gene dosage and genetic background are factors that influence transgene expression in transgenic plants. In our previous studies, we produced transgenic tomato plants that accumulate miraculin, a taste-modifying protein, in a genetically stable manner. To elucidate the effects of gene dosage and genetic background on miraculin accumulation in transgenic tomato fruits, we generated hybrid tomato lines between the homozygous transgenic line 56B (background cultivar 'Moneymaker') and the pure cultivars 'Micro-Tom,' 'Moneymaker,' 'Ailsa Craig,' 'M82,' 'Rutgers' and 'Aichi-first' and analyzed them for miraculin mRNA expression and miraculin protein accumulation. The hybrid lines exhibited variation in their fruit structures. Among the hybrid lines heterozygous for the miraculin gene, miraculin accumulation in the fruits varied from 111.0 mg g Ϫ1 fresh weight (FW) to 159.4 mg g Ϫ1 FW. Furthermore, the homozygous line 56B showed higher miraculin accumulation and miraculin mRNA expression than the heterozygous line 56Bϫ'Moneymaker.' These results demonstrate the profound effects of gene dosage and genetic background on miraculin accumulation in transgenic tomato fruits.Key words: Gene dosage, genetic background, miraculin, taste-modifying protein.Plant Biotechnology 27, 333-338 (2010) Original PaperThis article can be found at http://www.jspcmb.jp/ allelic composition and genetic background affect transgene expression and inheritance in white clover. Another study in rice showed that the expression pattern of Xa3/Xa26, which is a rice disease resistance gene, was affected by the genetic background (Cao et al. 2007). The full effects of gene dosage and genetic background on transgene expression and translation remain to be elucidated.In this study, we generated hybrid lines between transgenic line 56B (background cultivar 'Moneymaker'), which is homozygous for the miraculin gene, and the pure line cultivars 'Micro-Tom,' 'Moneymaker,' 'Ailsa Craig,' 'M82,' 'Rutgers' and 'Aichi-first.' Characterization of the hybrids' miraculin mRNA expression and miraculin protein accumulation, revealed the effects of gene dosage and genetic background on miraculin accumulation in transgenic tomato fruits, and in addition we discuss the significance of combining molecular breeding with cross-breeding to produce transgenic tomatoes that accumulate miraculin. Materials and methods Plant material and growth conditionsThe transgenic tomato line 56B was produced in our previous work (Sun et al. 2007). In brief miraculin encoding 660-bp DNA fragment was inserted into the XbaI/SacI sites of the plant transformation vectors pBI121 (Sun et al. 2006). Line 56B possesses a single copy of the miraculin gene driven by the CaMV 35S promoter.In the present study, the miraculin-accumulating transgenic tomato line 56B (background cultivars 'Moneymaker') was crossed with six other cultivars: 'Micro-Tom (accession number TOMJPF00001), ' 'Moneymaker (accession number TOMJPF00002), ' 'Aichi-first (accession number TOMJPF00003), ' 'Ailsa Cra...

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“…"Moneymaker"), which contain the miraculin gene (AB512278) driven by the CaMV 35S promoter-nos terminator cassette in the pBI121 vector and accumulate high concentrations of miraculin in the entire plant, have previously been produced and evaluated for their miraculin contents (Sun et al 2007;Yano et al 2010). The seeds of transgenic tomatoes and non-transgenic wild-type (WT) tomatoes were germinated on Petri dishes covered with moist filter paper at 25°C with a 16 h light/8 h dark cycle.…”

Section: Miraculin-accumulating Transgenic Tomatomentioning

confidence: 99%

Cultivation under salt stress increases the concentration of recombinant miraculin in transgenic tomato fruit, resulting in an increase in purification efficiency

Hirai

Duhita

Hiwasa-Tanase

et al. 2011

Plant Biotechnology

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Tomato is a suitable material for producing recombinant miraculin protein in genetically stable manner

Cited by 37 publications

References 19 publications

Vacuolar targeting of r‐proteins in sugarcane leads to higher levels of purifiable commercially equivalent recombinant proteins in cane juice

Vacuolar targeting of r‐proteins in sugarcane leads to higher levels of purifiable commercially equivalent recombinant proteins in cane juice

Gene dosage and genetic background affect miraculin accumulation in transgenic tomato fruits

Cultivation under salt stress increases the concentration of recombinant miraculin in transgenic tomato fruit, resulting in an increase in purification efficiency

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