Cyanobacterial Production of Biopharmaceutical and Biotherapeutic Proteins

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, CO 2 , and H 2 O.

Recombinant Protein Stability in Cyanobacteria

“…TEV. 20,22,27 In this case, the nptI gene itself was chosen as a leader sequence in the fusion construct, since it was shown to enhance the heterologous protein expression in cyanobacteria while, at the same time, acting as a selectable marker. 27 To this aim, fusion construct nptI*TEV (Figure 1C) was designed to replace the psbA2 gene in the respective locus.…”

“…Recent work from this lab showed that IFN, as a CpcB*IFN fusion construct, could accumulate in transformant cyanobacteria to about 12% of the total cellular protein. 20 However, it was not clear whether cleaved IFN from a CpcB*IFN fusion construct could be stable in the cyanobacterial cytosol. To address this question, the cpcB*IFN construct 20 was modified to install the cleavage tev sequence immediately prior to the IFN-encoding DNA (Figure 8A).…”

“…20 However, it was not clear whether cleaved IFN from a CpcB*IFN fusion construct could be stable in the cyanobacterial cytosol. To address this question, the cpcB*IFN construct 20 was modified to install the cleavage tev sequence immediately prior to the IFN-encoding DNA (Figure 8A).…”

“…20 Unfortunately, while the expression of heterologous prokaryotic proteins in cyanobacteria appears to be possible upon coupling a strong promoter and RBS to the DNA of interest, 21−23 the expression of eukaryotic proteins proved to be difficult, which is believed to be due to hindrances at the level of protein translation. 20,24 Overexpression of eukaryotic proteins in cyanobacteria was achieved indirectly upon implementing a "fusion constructs approach". This procedure comprised the fusion of a native or non-native gene that is highly expressed in cyanobacteria to the heterologous eukaryotic gene, with the first serving as a leader sequence.…”

Engineering microalgae: transition from empirical design to programmable cells

“…Biocontainment can be improved further by codon reassignment of the transgenes in the chloroplast [119]. Taken together, barriers encountered in the use of plantbased systems to produce recombinant protein or other heterologous products would make them less attractive than photosynthetic microorganisms, for example, microalgae and cyanobacteria [29]. More than 100 foreign or native proteins have thus far been produced in algal chloroplasts, including 40 different therapeutic proteins produced in C. reinhardtii chloroplasts [183].…”