Journal of Applied Phycology

, Volume 30, Issue 6, pp 3503–3518 | Cite as

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

  • Khawaja Muhammad Imran Bashir
  • Moo-Sang Kim
  • Ulf Stahl
  • Man-Gi ChoEmail author


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.


Chlorophyta Heterologous gene expression Hygromycin Microalgae Nuclear genome engineering Protein cell factory Recombinant protein 


Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Supplementary material

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ESM 1 (PDF 4649 kb)


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corrected publication December/2018

Authors and Affiliations

  1. 1.Department of Biotechnology, Division of Energy and BioengineeringDongseo UniversityBusanRepublic of Korea
  2. 2.Seafood Research Center, IACFSilla UniversityBusanRepublic of Korea
  3. 3.Institute of BiotechnologyTechnische Universität BerlinBerlinGermany

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