Abstract
Efforts to express human therapeutic proteins in photosynthetic organisms have been described in the literature. Regarding microalgae, most of the research entailed a heterologous transformation of the chloroplast, but transformant cells failed to accumulate the desired recombinant proteins in high quantity. The present work provides methods and DNA construct formulations for over-expressing in photosynthetic cyanobacteria, at the protein level, human-origin bio-pharmaceutical and bio-therapeutic proteins. Proof-of-concept evidence is provided for the design and reduction to practice of “fusion constructs as protein overexpression vectors” for the generation of the bio-therapeutic protein interferon alpha-2 (IFN). IFN is a member of the Type I interferon cytokine family, well-known for its antiviral and anti-proliferative functions. Fusion construct formulations enabled accumulation of IFN up to 12% of total cellular protein in soluble form. In addition, the work reports on the isolation and purification of the fusion IFN protein and preliminary verification of its antiviral activity. Combining the expression and purification protocols developed here, it is possible to produce fairly large quantities of interferon in these photosynthetic microorganisms, generated from sunlight, CO2, and H2O.
Highlights
Efforts to express human therapeutic proteins in photosynthetic microorganisms abound in the literature
Case study of this experimental work is the heterologous expression of the mature human interferon α-2 protein3, hereafter referred to as interferon alpha-2 (IFN), in the model cyanobacteria Synechocystis sp
The ∼27 kD protein could be the CpcG1 subunit of the phycobilisome, a phycocyanin rod-core linker polypeptide (Kondo et al, 2005), and the ∼108 kD band is tentatively attributed to a β-subunit of phycocyanin (CpcB)∗His∗IFN trimer, as it was shown to contain the CpcB∗His∗IFN fusion protein. These results suggest that α-subunit of phycocyanin (CpcA) and CpcG1 assemble with the CpcB∗His∗IFN, potentially as a CpcB∗His∗IFN-CpcA-CpcG1 complex, in a manner analogous to the α-β phycocyanin heterodimer assembly in the wild type, with the CpcG1 serving as a linker polypeptide, so that the complex binds to the column and elutes together from the resin upon application of 250 mM imidazole
Summary
Efforts to express human therapeutic proteins in photosynthetic microorganisms abound in the literature. In their preponderance, these entail heterologous transformation of microalgal chloroplasts as a synthetic biology platform for the production of biopharmaceutical and therapeutic proteins (Dyo and Purton, 2018, and references therein). The problem is exacerbated because of the frequent assumption in the field that a strong promoter will automatically cause gene overexpression when, in practice, SDS-PAGE fails to show presence of the transgenic protein and only sensitive Western blot analysis can offer evidence of low-levels of expression. A qualitative rule-of-thumb for overexpression in this respect is ability to detect the transgenic protein in SDS-PAGE analysis of total protein extracts
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