Designing a new generation of expression toolkits for engineering of green microalgae; robust production of human interleukin-2

Abstract

Introduction: Attributable to some critical features especially the similarity of the protein synthesis machinery between humans and microalgae, these microorganisms can be utilized for the expression of many recombinant proteins. However, low and unstable gene expression levels prevent the further development of microalgae biotechnology towards protein production. Methods: Here, we designed a novel "Gained Agrobacterium-2A plasmid for microalgae expression" (named GAME plasmid) for the production of the human interleukin-2 using three model microalgae, including Chlamydomonas reinhardtii, Chlorella vulgaris, and Dunaliella salina. The GAME plasmid harbors a native chimeric hsp70/Int-1/rbcS2 promoter, the microalgae specific Kozak sequence, a novel hybrid 2A peptide, and Int-1 and Int-3 of the rbcS2 gene in its expression cassette. Results: The obtained data confirmed that the GAME plasmid can transform the microalgae with high transformation frequency. Molecular and proteomic analyses revealed the stable and robust production of the hIL-2 by the GAME plasmid in the microalgae. According to the densimetric analysis, the microalgae can accumulate the produced protein about 0.94% of the total soluble protein content. The ELISA data confirmed that the produced hIL-2 possesses the same conformation pattern with the acceptable biological activity found naturally in humans. Conclusion: Most therapeutic proteins need post-translational modifications for their correct conformation, biological function, and half-life. Accordingly, microalgae could be considered as a cost-effective and more powerful platform for the production of a wide range of recombinant proteins such as antibodies, enzymes, hormones, and vaccines.

Microalgae as an Efficient Vehicle for the Production and Targeted Delivery of Therapeutic Glycoproteins against SARS-CoV-2 Variants

“…Microalgae are unicellular photosynthetic eukaryotic organisms that have recently been identified as an efficient and environmentally-friendly alternative for the production of recombinant proteins, including complex glycoproteins [64][65][66][67][68]. Microalgae are able to properly impose post-translational modifications such as glycosylation and disulfide bridges on recombinant proteins [69].…”

“…Due to their rapid growth rate, simple and inexpensive media, and the ability to use autotrophic culture, microalgae represent a cost-effective platform for the production of recombinant ACE2 and S-glycoproteins [64,71,81]. More importantly, eukaryotic microalgae can correctly impose post-translational modification with high homogeneity on recombinant glycoproteins [67,101].…”

Advanced Applications for Protein and Compounds from Microalgae

“…Microalgae represent an important biotechnology resource for the production of recombinant proteins for pharmacological applications. Successful results were reported by the genetic manipulation of microalgae for the production of antigens for vaccines, antibodies, immunotoxins, hormones, and antimicrobial agents [ 59 , 60 , 61 , 62 , 63 , 64 , 65 , 66 , 67 , 68 , 69 , 70 , 71 , 72 , 73 , 74 , 75 , 76 , 77 , 78 , 79 , 80 , 81 , 82 , 83 , 84 , 85 , 86 , 87 ]. Strategies and techniques have been developed for the exploitation of microalgae as expression systems for a number of advanced genetic toolkits [ 62 , 63 , 64 , 65 ].…”

“…On the other hand, nuclear transformation produced a lower protein accumulation, but retained PTMs, such as N-glycosylation [ 59 , 67 ]. Furthermore, nuclear manipulation provided tools for the targeting of proteins into specific subcellular compartments or secreting them into culture media [ 72 ] while regulatory elements (promoters and terminators) and selectable markers were tested to identify improved strategies for higher levels of recombinant protein [ 15 , 73 ].…”

“…Antigens, antibiotics, and other bioactive molecules against animal pathogens have been produced. Examples include swine fever virus [ 74 ], white spot syndrome virus [ 64 , 72 , 73 ], lethal shrimp yellow head virus [ 77 , 78 ], Aeromonas hydrophila bacteria [ 79 ], and vibriosis disease [ 80 ].…”