Functional Improvement of Human Cardiotrophin 1 Produced in Tobacco Chloroplasts by Co-Expression with Plastid Thioredoxin m

María Ancín,Jon Veramendi,Ruth Sanz-Barrio,Luis Larraya,Inmaculada Farran,Alicia Fernández-San Millán,Eva Santamaría

doi:10.3390/plants9020183

María Ancín, Jon Veramendi + Show 5 more

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https://doi.org/10.3390/plants9020183

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Abstract

Human cardiotrophin 1 (CT1), a cytokine with excellent therapeutic potential, was previously expressed in tobacco chloroplasts. However, the growth conditions required to reach the highest expression levels resulted in an impairment of its bioactivity. In the present study, we have examined new strategies to modulate the expression of this recombinant protein in chloroplasts so as to enhance its production and bioactivity. In particular, we assessed the effect of both the fusion and co-expression of Trx m with CT1 on the production of a functional CT1 by using plastid transformation. Our data revealed that the Trx m fusion strategy was useful to increase the expression levels of CT1 inside the chloroplasts, although CT1 bioactivity was significantly impaired, and this was likely due to steric hindrance between both proteins. By contrast, the expression of functional CT1 was increased when co-expressed with Trx m, because we demonstrated that recombinant CT1 was functionally active during an in vitro signaling assay. While Trx m/CT1 co-expression did not increase the amount of CT1 in young leaves, our results revealed an increase in CT1 protein stability as the leaves aged in this genotype, which also improved the recombinant protein’s overall production. This strategy might be useful to produce other functional biopharmaceuticals in chloroplasts.

Highlights

Proteins are widely used in medicine as diagnostic reagents, vaccines and drugs, and this creates a strong demand for the production of recombinant proteins on an industrial scale.Commercial protein production has traditionally relied on microbial fermentation and mammalian cell lines
The fusion gene was expressed from the tobacco psbA promoter and 50 -UTR regulatory sequences, which allowed very high levels of recombinant proteins to be expressed in chloroplasts [33,34]
Human cardiotrophin 1 (CT1) was previously expressed in tobacco chloroplasts at relatively high levels [14], their functionality was impaired when plants were grown under high-light conditions, most likely due to improper protein folding

Summary

Introduction

Proteins are widely used in medicine as diagnostic reagents, vaccines and drugs, and this creates a strong demand for the production of recombinant proteins on an industrial scale.Commercial protein production has traditionally relied on microbial fermentation and mammalian cell lines. Proteins are widely used in medicine as diagnostic reagents, vaccines and drugs, and this creates a strong demand for the production of recombinant proteins on an industrial scale. The use of plants as bioreactors, a technology known as plant molecular farming, offers several advantages over traditional systems such as reduced manufacturing costs, minimized risk of contamination with human pathogens or toxins, and production that is scalable [1]. Transgenic plants are potentially one of the most economical systems for large-scale production of recombinant proteins for industrial and pharmaceutical uses [2]. Several additional advantages to chloroplast technology can be noted, the maternal inheritance of plastids and their DNA [7], which minimizes outcrossing of transgenic pollen with related weeds or crops, and increases the biosafety of genetically modified plants. The main problem of plant molecular farming is the high cost of purifying the recombinant proteins

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