Over‐Expression of a Phycocyanin‐Interferon Fusion and Differential Cleaving Efficiency in Cyanobacteria ( <i>Synechocystis</i> sp. PCC 6803)

ABSTRACT Recombinant protein expression in heterologous biological systems is an expanding field in synthetic biology. Photosynthetic organisms have the potential to provide an efficient low‐cost platform for recombinant protein production because they require minimal growth nutrients and are less susceptible to zoonotic and other contaminants. Cyanobacteria are a class of microorganisms that are gaining as the preferred photosynthetic cell factories for product generation. In this study, the cyanobacterium Synechocystis sp. PCC 6803 was used as a host to stably over‐express a functional form of the human interferon α−2 (IFN), as a fusion construct with the abundant CpcB β‐subunit of phycocyanin. To cleave and isolate the free form of IFN from the fusion protein, different constructs were designed containing the Tobacco Etch Virus (TEV) or Human Rhinovirus (hrv) 3 C protease cleaving loci, placed between the leading CpcB and trailing IFN moieties of the fusion proteins. The work examined the comparative cleaving efficacy of TEV and HRV proteases in separating IFN from such over‐expressed phycocyanin fusion protein complexes. It was concluded that the HRV protease system is superior to that of TEV, and that of other cleaving proteases recently tested, and may thus be incorporated in the toolkit of cyanobacterial synthetic biology for recombinant protein synthesis and isolation.