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Agrobacterium-mediated genetic transformation of Dictyosphaerium pulchellum for the expression of erythropoietin

Bashir, Khawaja Muhammad Imran, Kim, Moo-Sang, Stahl, Ulf, Cho, Man-Gi
Journal of applied phycology 2018 v.30 no.6 pp. 3503-3518
Agrobacterium radiobacter, Dictyosphaerium, Western blotting, acetosyringone, affinity chromatography, biopharmaceuticals, coculture, erythropoietin, freshwater, gene expression, genetic engineering, genetic transformation, genetic vectors, heterologous gene expression, metabolic engineering, microalgae, monoclonal antibodies, nuclear genome, photons, polyacrylamide gel electrophoresis, protein synthesis, quantitative polymerase chain reaction, recombinant proteins, resistance genes, selective media, transfer DNA, transgenes
Recombinant proteins are extensively used for a growing number of fields in biology. However, microalgal species have not been widely adopted as cell factories for recombinant protein production. Unique metabolic properties, ease of cultivation, fast growth rates, and continuous progress in genetic engineering of microalgae have raised interest in the use of microalgae species for recombinant protein production. Here, we report an Agrobacterium-mediated genetic transformation system for the heterologous expression of a therapeutic protein, “erythropoietin,” in a nonmodel green microalga, Dictyosphaerium pulchellum. Hygromycin resistance gene (Hyg) was used as a selectable marker. The genetic transformation of D. pulchellum was performed in modified AF6 medium supplemented with 150 μM acetosyringone, co-cultivated for 48 h at 25 ± 2 °C and a light intensity of 18 ± 2 μmol photons m⁻² s⁻¹. Co-cultivation of D. pulchellum with Agrobacterium tumefaciens harboring the binary expression vector pCAMBIA1301-Hyg-EPO-Hisₜₐg yielded hygromycin-resistant colonies on selective medium after 2–3 weeks. Gene integration into the D. pulchellum nuclear genome was confirmed by PCR amplification of T-DNA from the genomic DNA of hygromycin-resistant and wild-type strains. Interestingly, SDS-PAGE and subsequent Western blotting with His-tag monoclonal and anti-erythropoietin monoclonal antibodies revealed an EPO-specific signal slightly below 34 kDa. Furthermore, EPO gene expression and transgene copy numbers were estimated by quantitative real-time PCR. Approximately, 500 μg L⁻¹ of extracellular recombinant erythropoietin protein was purified by His-tag affinity chromatography. The developed genetic transformation system would allow the metabolic engineering and a better alternative to produce recombinant therapeutic proteins from nonmodel freshwater microalgae species.