Evaluation of lettuce chloroplast and soybean cotyledon as platforms for production of functional bone morphogenetic protein 2

Queiroz, Lídia N.; Maldaner, Franciele Roberta; Mendes, Érica Araújo; Sousa, Aline R.; Dall’Astta, Rebeca C.; Mendonça, Gustavo; Mendonça, Daniela Baccelli Silveira; Aragão, F. J. L.

doi:10.1007/s11248-019-00116-7

Cited by 14 publications

(8 citation statements)

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“…To date, to the best of our knowledge, the successful expression of the mature form of human BMP2 using plant systems has not been reported. Previous attempts to introduce hBMP2m DNA into the lettuce chloroplast genome did not result in significant accumulation of hBMP2m (Queiroz et al 2019 ). Other studies produced hBMP2m by fusing it with GUS and zein at the N-terminus, but the hBMP2m recombinant protein yield in tobacco leaves dropped to a mere 0.02% TSP after removing the fused domains (Ceresoli et al 2016 ; Suo et al 2006 ).…”

Section: Discussionmentioning

confidence: 97%

“…Various plant-based production systems, such as tobacco leaves and soybean seeds, have successfully produced hBMP2 (Ceresoli et al 2016 ; Queiroz et al 2019 ; Rahimifard Hamedani et al 2021 ; Suo et al 2006 ). However, these systems typically only produce pro-hBMP2 or β-glucuronidase-fused, γ-zein-fused, or hydrophobin-fused hBMP2, and yield of the mature hBMP2 (hBMP2m) in current plant systems is low.…”

Section: Introductionmentioning

confidence: 99%

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High-yield BMP2 expression in rice cells via CRISPR and endogenous αAmy3 promoter

Nguyen,

Wu,

Chang

et al. 2024

Appl Microbiol Biotechnol

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Plant cells serve as versatile platforms for the production of high-value recombinant proteins. This study explored the efficacy of utilizing an endogenous αAmy3 promoter for the expression of a bioactive pharmaceutical protein, specifically the mature region of human bone morphogenetic protein 2 (hBMP2m). Utilizing a refined CRISPR/Cas9-mediated intron-targeting insertion technique, which incorporates an artificial 3’ splicing site upstream of the target gene, we achieved a transformation efficiency of 13.5% in rice calli that carried the rice-codon optimized mature region of hBMP2 cDNA (rhBMP2m) in the αAmy3 intron 1. Both homozygous and heterozygous rhBMP2m knock-in rice suspension cell lines were generated. These lines demonstrated the endogenous αAmy3 promoter regulated rhBMP2m mRNA and rhBMP2m recombinant protein expression, with strongly upregulation in respond to sugar depletion. The homozygous rhBMP2m knock-in cell line yielded an impressive 21.5 μg/mL of rhBMP2m recombinant protein, accounting for 1.03% of the total soluble protein. The high-yield expression was stably maintained across two generations, indicating the genetic stability of rhBMP2m gene knock-in at the αAmy3 intron 1 locus. Additionally, the rice cell-derived rhBMP2m proteins were found to be glycosylated, capable of dimer formation, and bioactive. Our results indicate that the endogenous rice αAmy3 promoter–signal peptide-based expression system is an effective strategy for producing bioactive pharmaceutical proteins. Key points • The endogenous αAmy3 promoter-based expression system enhanced the yield of BMP2 • The increased yield of BMP2 accounted for 1.03% of the total rice-soluble proteins • The rice-produced BMP2 showed glycosylation modifications, dimer formation, and bioactivity

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Section: Discussionmentioning

confidence: 97%

Section: Introductionmentioning

confidence: 99%

High-yield BMP2 expression in rice cells via CRISPR and endogenous αAmy3 promoter

Nguyen,

Wu,

Chang

et al. 2024

Appl Microbiol Biotechnol

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“…Independent from that, we produced an intact, bioactive FGF21-F-Tf fusion protein, accumulating to 6.7 and 5.6 mg kg –1 in transgenic tobacco seeds and leaves respectively, whereas transient expression in N. benthamiana leaves generated up to 2.1 mg kg –1 . The yield of FGF21-F-Tf in seeds was 22-fold higher compared to recombinant IL6 for which the constitutive 35S CaMV expression cassette has been used as well but substantially lower compared to other proteins that were expressed with a seed-specific promotor/terminator in tobacco seeds at accumulation levels of 190−6,500 mg kg –1 SDM ( Supplementary Table 6 ; Fiedler and Conrad, 1995 ; Kusnadi et al, 1998 ; De Jaeger et al, 2002 ; Cheung et al, 2009 ; Zimmermann et al, 2009 ; Zhang et al, 2010 ; Nausch et al, 2012b ; Hensel et al, 2015 ; Hernandez-Velazquez et al, 2015 ; Weichert et al, 2016 ; Ceballo et al, 2017 ; Queiroz et al, 2019 ). Therefore, using a seed-specific expression cassette might increase FGF21-Tf fusion protein yields in the future.…”

Section: Discussionmentioning

confidence: 99%

Seed- and leaf-based expression of FGF21-transferrin fusion proteins for oral delivery and treatment of non-alcoholic steatohepatitis

Hou

Bishop²,

Huckauf³

et al. 2022

Front. Plant Sci.

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Non-alcoholic steatohepatitis (NASH) is a global disease with no effective medication. The fibroblast growth factor 21 (FGF21) can reverse this liver dysfunction, but requires targeted delivery to the liver, which can be achieved via oral administration. Therefore, we fused FGF21 to transferrin (Tf) via a furin cleavage site (F), to promote uptake from the intestine into the portal vein, yielding FGF21-F-Tf, and established its production in both seeds and leaves of commercial Nicotiana tabacum cultivars, compared their expression profile and tested the bioavailability and bioactivity in feeding studies. Since biopharmaceuticals need to be produced in a contained environment, e.g., greenhouses in case of plants, the seed production was increased in this setting from 239 to 380 g m–2 a–1 seed mass with costs of 1.64 € g–1 by side branch induction, whereas leaves yielded 8,193 g m–2 a–1 leave mass at 0.19 € g–1. FGF21-F-Tf expression in transgenic seeds and leaves yielded 6.7 and 5.6 mg kg–1 intact fusion protein, but also 4.5 and 2.3 mg kg–1 additional Tf degradation products. Removing the furin site and introducing the liver-targeting peptide PLUS doubled accumulation of intact FGF21-transferrin fusion protein when transiently expressed in Nicotiana benthamiana from 0.8 to 1.6 mg kg–1, whereas truncation of transferrin (nTf338) and reversing the order of FGF21 and nTf338 increased the accumulation to 2.1 mg kg–1 and decreased the degradation products to 7% for nTf338-FGF21-PLUS. Application of partially purified nTf338-FGF21-PLUS to FGF21–/– mice by oral gavage proved its transfer from the intestine into the blood circulation and acutely affected hepatic mRNA expression. Hence, the medication of NASH via oral delivery of nTf338-FGF21-PLUS containing plants seems possible.

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“…Verônica, cv Flora and cv. Cisco [71][72][73] Plantaginaceae Scoparia dulcis [74] Cucurbitaceae Momordica charantia [75] Amaranthaceae Beta vulgaris [76] Salicaceae Populus alba [77,78] Monocots Poaceae Saccharum officinarum [79] Oryza sativa var. Japonica line 19 and Hwa-Chung [80,81] Liverworts Marchantiaceae Marchantia polymorpha [82] Moss Funariaceae Physcomitrella patens [83] Algae Microalgae Isochrysidaceae Tisochrysis lutea [84] Chloroplasts: The Future of Large-Scale Protein Production DOI: http://dx.doi.org/10.5772/intechopen.111829 proteins for vaccine production, proteins of pharmaceutical interest, and proteins for the production of biofuels and biomaterials [36, [94][95][96][97], as well as proteins that confer resistance to pests and diseases [77,98,99].…”

Section: Incorporation Of New Traits In Plants Through Chloroplast Tr...mentioning

confidence: 99%

Chloroplasts: The Future of Large-Scale Protein Production

Chávez¹,

Ornelas²,

Cruz³

et al. 2024

Physiology

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Chloroplast engineering has matured considerably in recent years. It is emerging as a promising tool to address the challenges related to food security, drug production, and sustainable energy posed by an ever-growing world population. Chloroplasts have proven their potential by efficiently expressing transgenes, encapsulating recombinant proteins, and protecting them from cellular machinery, making it possible to obtain highly functional proteins. This quality has also been exploited by interfering RNA technology. In addition to the practical attributes offered by chloroplast transformation, such as the elimination of position effects, polycistronic expression, and massive protein production, the technique represents an advance in biosafety terms; however, even if its great biotechnological potential, crops that have efficiently transformed are still a proof of concept. Despite efforts, other essential crops have remained recalcitrant to chloroplast transformation, which has limited their expansion. In this chapter, we address the most recent advances in this area and the challenges that must be solved to extend the transformation to other crops and become the de facto tool in plant biotechnology.

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Evaluation of lettuce chloroplast and soybean cotyledon as platforms for production of functional bone morphogenetic protein 2

Cited by 14 publications

References 58 publications

High-yield BMP2 expression in rice cells via CRISPR and endogenous αAmy3 promoter

High-yield BMP2 expression in rice cells via CRISPR and endogenous αAmy3 promoter

Seed- and leaf-based expression of FGF21-transferrin fusion proteins for oral delivery and treatment of non-alcoholic steatohepatitis

Chloroplasts: The Future of Large-Scale Protein Production

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