Stable Expression of Basic Fibroblast Growth Factor in Chloroplasts of Tobacco

Wang, Yunpeng; Wei, Zhengyi; Zhong, Xiaofang; Lin, Chung-Hung; Cai, Yuhong; Ma, Jing; Zhang, Yuying; Liu, Yanzhi; Xing, Shaochen

doi:10.3390/ijms17010019

Cited by 27 publications

(19 citation statements)

References 42 publications

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“…In vitro assays of inserted genes with several synonymous codons show that translation efficiency does not always correlate with codon usage in plastid mRNAs (Nakamura and Sugiura, 2007), but they have been used in several codon optimization studies (Lutz et al, 2001;Ye et al, 2001;Franklin et al, 2002;Lenzi et al, 2008;Jabeen at al., 2010;Madesis et al, 2010;Gisby et al, 2011;Wang et al, 2015b;Boehm et al, 2016;Nakamura et al, 2016). While some studies achieved significant increases in expression (75-to 80-fold) after codon optimization (Franklin et al, 2002;Gisby et al, 2011), other studies observed negligible enhancement (Ye et al, 2001;Lenzi et al, 2008;Daniell et al, 2009;Wang et al, 2015b;Nakamura et al, 2016).…”

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confidence: 99%

Codon Optimization to Enhance Expression Yields Insights into Chloroplast Translation

et al. 2016

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Codon optimization based on psbA genes from 133 plant species eliminated 105 (human clotting factor VIII heavy chain [FVIII HC]) and 59 (polio VIRAL CAPSID PROTEIN1 [VP1]) rare codons; replacement with only the most highly preferred codons decreased transgene expression (77-to 111-fold) when compared with the codon usage hierarchy of the psbA genes. Targeted proteomic quantification by parallel reaction monitoring analysis showed 4.9-to 7.1-fold or 22.5-to 28.1-fold increase in FVIII or VP1 codon-optimized genes when normalized with stable isotope-labeled standard peptides (or housekeeping protein peptides), but quantitation using western blots showed 6.3-to 8-fold or 91-to 125-fold increase of transgene expression from the same batch of materials, due to limitations in quantitative protein transfer, denaturation, solubility, or stability. Parallel reaction monitoring, to our knowledge validated here for the first time for in planta quantitation of biopharmaceuticals, is especially useful for insoluble or multimeric proteins required for oral drug delivery. Northern blots confirmed that the increase of codonoptimized protein synthesis is at the translational level rather than any impact on transcript abundance. Ribosome footprints did not increase proportionately with VP1 translation or even decreased after FVIII codon optimization but is useful in diagnosing additional rate-limiting steps. A major ribosome pause at CTC leucine codons in the native gene of FVIII HC was eliminated upon codon optimization. Ribosome stalls observed at clusters of serine codons in the codon-optimized VP1 gene provide an opportunity for further optimization. In addition to increasing our understanding of chloroplast translation, these new tools should help to advance this concept toward human clinical studies.

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confidence: 99%

Codon Optimization to Enhance Expression Yields Insights into Chloroplast Translation

et al. 2016

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“…The vector pWYP21406 was transformed into the tobacco chloroplasts by using the biolistic particle bombardment as described previously 40 . The leaves of one-month-old tobacco seedlings were used as explants, the plasmid pWYP21406 was coated with 0.66 µm gold particles, and the bombardment was carried out at 1100 psi with a shooting distance of 6 cm and a vacuum of 28 on MS medium.…”

Section: Chloroplast Transformation and Generation Of Transplastomic ...mentioning

confidence: 99%

“…However, expression of hFGF21 in plant chloroplast is not yet reported, although a member of the FGFs family, bFGF (FGF2) has been demonstrated to be successfully expressed in tobacco chloroplasts 40 . In this paper, the successful expression of hFGF21 fused to GFP at a high level (12.44 ± 0.45% of TSP) in tobacco chloroplasts is rstly reported.…”

Section: Introductionmentioning

confidence: 99%

Hyper Expression of Active, GFP-fused HFGF21 in Tobacco Chloroplasts

Wang

Fan

et al. 2022

Preprint

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Human fibroblast growth factor 21 (hFGF21) is a promising candidate for metabolic diseases. In this study, a tobacco chloroplast transformation vector, pWYP21406, was constructed. In the vector, the codon optimized encoding gene hFGF21 fused with GFP at its 5’ terminal was driven by the promoter of plastid rRNA operon (Prrn) and terminated by the terminator of plastid rps16 gene (Trps16). Spectinomycin resistant gene (aadA) as the selectable marker was placed in the same cistron between hFGF21 and the terminator Trps16. Transplastomic plants were generated by biolistic bombardment method and proven to be homoplastic by Southern Blotting analyses. The expression of GFP was detected under UV light and laser confocal microscope. The expression of GFP-hFGF21 was confirmed by immunoblotting and quantified by ELISA. The accumulation of was GFP-hFGF21confirmed to be 12.44 ± 0.45% of total soluble protein (i.e., 1.9232 ± 0.0673 g kg− 1 of fresh weight). The GFP-hFGF21 was confirmed to be capable of promoting the proliferation of hepatoma cell line HepG2, inducing the expression of glucose transporter 1 in hepatoma HepG2 cells and improve the glucose uptakes. The above results suggested that chloroplast expression is a promising approach for bioactive recombinant hFGF21 production.

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“…Basic fibroblast growth factor (bFGF) accelerates cell proliferation and differentiation and hence possesses wide clinical applications. A codon-optimized bFGF gene was transformed to tobacco chloroplasts, and recombinant protein accumulation was observed (Wang et al 2015). Serum antibodies developed in hemophilia B patients against coagulation factor IX (FIX) is highly challenging to eliminate due to nephrotic syndrome after continued infusion.…”

Section: Plastid Bioreactors For Molecular Farmingmentioning

confidence: 99%

Green Biotechnology: A Brief Update on Plastid Genome Engineering

Bharadwaj¹,

Kumar

Ramalingam

2019

Advances in Plant Transgenics: Methods and Applications

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Plant genetic engineering has become an inevitable tool in the molecular breeding of crops. Significant progress has been made in the generation of novel plastid transformation vectors and optimized transformation protocols. There are several advantages of plastid genome engineering over conventional nuclear transformation. Some of the advantages include multigene engineering by expression of biosynthetic pathway genes as operons, extremely high-level expression of protein accumulation, lack of transgene silencing, etc. Transgene containment owing to maternal inheritance is another important advantage of plastid genome engineering. Chloroplast genome modification usually results in alteration of several thousand plastid genome copies in a cell. Several therapeutic proteins, edible vaccines, antimicrobial peptides, and industrially important enzymes have been successfully expressed in chloroplasts so far. Here, we critically recapitulate the latest developments in plastid genome engineering. Latest advancements in plastid genome sequencing are briefed. In addition, advancement of extending the toolbox for plastid engineering for selected applications in the area of molecular farming and production of industrially important enzyme is briefed.

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Stable Expression of Basic Fibroblast Growth Factor in Chloroplasts of Tobacco

Cited by 27 publications

References 42 publications

Codon Optimization to Enhance Expression Yields Insights into Chloroplast Translation

Codon Optimization to Enhance Expression Yields Insights into Chloroplast Translation

Hyper Expression of Active, GFP-fused HFGF21 in Tobacco Chloroplasts

Green Biotechnology: A Brief Update on Plastid Genome Engineering

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