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Production of biologically active human basic fibroblast growth factor (hFGFb) using Nicotiana tabacum transplastomic plants

June 2024
Planta 260(1)

June 2024
260(1)

DOI:10.1007/s00425-024-04456-5

Authors:

Carolina Müller

National Scientific and Technical Research Council

Nicolás Budnik

IBioBA - CONICET - Partner Institute of the Max Planck Society

Show all 8 authorsHide

Main conclusion We generated transplastomic tobacco lines that stably express a human Basic Fibroblast Growth Factor (hFGFb) in their chloroplasts stroma and purified a biologically active recombinant hFGFb. Main The use of plants as biofactories presents as an attractive technology with the potential to efficiently produce high-value human recombinant proteins in a cost-effective manner. Plastid genome transformation stands out for its possibility to accumulate recombinant proteins at elevated levels. Of particular interest are recombinant growth factors, given their applications in animal cell culture and regenerative medicine. In this study, we produced recombinant human Fibroblast Growth Factor (rhFGFb), a crucial protein required for animal cell culture, in tobacco chloroplasts. We successfully generated two independent transplastomic lines that are homoplasmic and accumulate rhFGFb in their leaves. Furthermore, the produced rhFGFb demonstrated its biological activity by inducing proliferation in HEK293T cell lines. These results collectively underscore plastid genome transformation as a promising plant-based bioreactor for rhFGFb production.

Generation of transplastomic plants for production of rhFGFb. a pUTR-rhFGFb vector containing the recombinogenic sequences flanking the selective gene (aadA) and the transgene (hFGFb). Left flanking region (LFR): 3’ region of the 16S ribosomal RNA sequence (rrn16); Right flanking region (RFR): trnI and 5’ region of trnA genes. The rhFGFb transgene includes an open reading frame encoding a N-terminal Poly HIS-tag (6xHIS) followed by the recognition site of Tobacco Etch Virus protease (TEV) and the coding region of human FGFb. b Schematic representation of the integration site in the plastome. Transgenes are integrated in the intergenic region between rrn16 and trnI (grey arrow). c Phenotype of transplastomic plants. Pictures of representative plants for each transplastomic line and the NT control taken after 5 and 9 weeks of germination

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Analysis of homoplasmy and transgene inheritance. a Southern blot analysis (upper) and schematic representation of transformed and non-transformed cpDNA (lower) showing the restriction enzyme site and the trnI/A probe used for DNA hybridization. UTR-GUS non-related transplastomic plant, NT non-transfomed N. tabacum, UTR-FGF transplastomic lines. b Germination assay. 100 seeds from each line were germinated in MS medium (MS) with or without spectinomycin (SP). Pictures were taken after 7 days

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Analysis of protein accumulation in UTR-FGF transplastomic lines. Western blot analysis using anti-HIS antibody of rhFGFb accumulation in UTR-FGF lines (a) and rhFGFb accumulation in leaves (b) from different developmental stages. The different leaves used for protein extraction are marked with numbers starting from the first fully unfolded leaf (b, right). The RuBisCO large subunit (Rb-L) was used as a loading control (ponceau red dyed)

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Purification of rhFGFb. a Schematic representation of the purification protocol. b, c Purification profile. Protein fractions from the purification protocol were analyzed by SDS-PAGE and coomasie blue staining (b) or western blot with anti-HIS antibody (c). d Key fractions from the purification protocol were analyzed by western blot with anti-HIS (left) or anti-FGF antibody (right). CS concentrated sample, Per percolate, E1-E4 elution fractions, - empty lane, NT E3 elution fraction from NT plant

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Proliferation assay in HEK293T cells. Rezazurin proliferation assay of HEK293T cells at 24 h post-induction with non-transformed plant extract (NT), purified plant rhFGFb or commercial rhFGFb (Gibco, PHG0026). Comparison of means of NT control vs rhFGFb (P = 0.0182) or commercial rhFGFb, PHG0026 (P = 0.0269) by one-way ANOVA (n = 3)

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Vol.:(0123456789)

Planta (2024) 260:28

https://doi.org/10.1007/s00425-024-04456-5

ORIGINAL ARTICLE

Production ofbiologically active human basic ﬁbroblast growth factor

(hFGFb) using Nicotiana tabacum transplastomic plants

CarolinaMüller1· NicolásBudnik2· FedericoGabrielMirkin1· CatalinaFranciscaVater1·

FernandoFélixBravo‑Almonacid1,3· CarolinaPerez‑Castro2· SoniaAlejandraWirth4,5· MaríaEugeniaSegretin1,6

Received: 6 January 2024 / Accepted: 2 June 2024

Abstract

Main conclusion We generated transplastomic tobacco lines that stably express a human Basic Fibroblast Growth

Factor (hFGFb) in their chloroplasts stroma and puriﬁed a biologically active recombinant hFGFb.

Main The use of plants as biofactories presents as an attractive technology with the potential to eﬃciently produce high-

value human recombinant proteins in a cost-eﬀective manner. Plastid genome transformation stands out for its possibility to

accumulate recombinant proteins at elevated levels. Of particular interest are recombinant growth factors, given their appli-

cations in animal cell culture and regenerative medicine. In this study, we produced recombinant human Fibroblast Growth

Factor (rhFGFb), a crucial protein required for animal cell culture, in tobacco chloroplasts. We successfully generated two

independent transplastomic lines that are homoplasmic and accumulate rhFGFb in their leaves. Furthermore, the produced

rhFGFb demonstrated its biological activity by inducing proliferation in HEK293T cell lines. These results collectively

underscore plastid genome transformation as a promising plant-based bioreactor for rhFGFb production.

Keywords Basic ﬁbroblast growth factor· Cell culture· Molecular farming· Plant-based bioreactors· Plastid

transformation· Tobacco

Abbreviations

hFGFb Human basic ﬁbroblast growth factor

rhFGFb Recombinant human ﬁbroblast growth factor

NT Non-transformed

TSP Total soluble protein

Introduction

The use of plant-based bioreactors stands as an attrac-

tive method for the production of recombinant proteins

for multiple applications (Tschofen etal. 2016). Plant

molecular farming oﬀers the advantage of cost-eﬀective

and large-scale production of heterologous proteins lever-

aging the abundance of plant biomass and the feasibility of

utilizing established agricultural procedures for scaling-up

Communicated by Dorothea Bartels.

* María Eugenia Segretin

segretin@dna.uba.ar

1 Instituto de Investigaciones en Ingeniería Genética

y Biología Molecular “Dr. Héctor N. Torres”

(INGEBI-CONICET), Vuelta de Obligado 2490,

CiudadAutónomaBuenosAiresC1428ADN, Argentina

2 Instituto de Investigación en Biomedicina de Buenos

Aires (IBioBA-CONICET)- Partner Institute

oftheMax Planck Society, Godoy Cruz 2390,

CiudadAutónomaBuenosAiresC1425FQ, Argentina

3 Departamento de Ciencia y Tecnología, Universidad

Nacional de Quilmes, Roque Sáenz Peña 352, Bernal,

BuenosAiresB1876BXD, Argentina

4 Instituto de Biodiversidad y Biología Experimental y

Aplicada (IBBEA-CONICET-UBA), Intendente Güiraldes

2160, CiudadAutónomaBuenosAiresC1428EGA,

Argentina

5 Laboratorio de Agrobiotecnología, Departamento de

Fisiología, Biología Molecular y Celular, Facultad

de Ciencias Exactas y Naturales, Universidad

de Buenos Aires, Intendente Güiraldes 2160,

CiudadAutónomaBuenosAiresC1428EGA, Argentina

6 Departamento de Fisiología, Biología Molecular y

Celular, Facultad de Ciencias Exactas y Naturales,

Universidad de Buenos Aires, Intendente Güiraldes 2160,

CiudadAutónomaBuenosAiresC1428EGA, Argentina

Content courtesy of Springer Nature, terms of use apply. Rights reserved.

ResearchGate has not been able to resolve any citations for this publication.

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Production of biologically active human basic fibroblast growth factor (hFGFb) using Nicotiana tabacum transplastomic plants

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