Selenocystamine improves protein accumulation in chloroplasts of eukaryotic green algae

Abstract

Eukaryotic green algae have become an increasingly popular platform for recombinant proteins production. In particular, Chlamydomonas reinhardtii, has garnered increased attention for having the necessary biochemical machinery to produce vaccines, human antibodies and next generation cancer targeting immunotoxins. While it has been shown that chloroplasts contain chaperones, peptidyl prolylisomerases and protein disulfide isomerases that facilitate these complex proteins folding and assembly, little has been done to determine which processes serve as rate-limiting steps for protein accumulation. In other expression systems, as Escherichia coli, Chinese hamster ovary cells, and insect cells, recombinant protein accumulation can be hampered by cell’s inability to fold the target polypeptide into the native state, resulting in aggregation and degradation. To determine if chloroplasts’ ability to oxidize proteins that require disulfide bonds into a stable conformation is a rate-limiting step of protein accumulation, three recombinant strains, each expressing a different recombinant protein, were analyzed. These recombinant proteins included fluorescent GFP, a reporter containing no disulfide bonds; Gaussia princeps luciferase, a luminescent reporter containing disulfide bonds; and an immunotoxin, an antibody-fusion protein containing disulfide bonds. Each strain was analyzed for its ability to accumulate proteins when supplemented with selenocystamine, a small molecule capable of catalyzing the formation of disulfide bonds. Selenocystamine supplementation led to an increase in luciferase and immunotoxin but not GFP accumulation. These results demonstrated that selenocystamine can increase the accumulation of proteins containing disulfide bonds and suggests that a rate-limiting step in chloroplast protein accumulation is the disulfide bonds formation in recombinant proteins native structure.

New tools for chloroplast genetic engineering allow the synthesis of human growth hormone in the green alga Chlamydomonas reinhardtii

“…This mutation blocks the trans -splicing of psaA-exon1 mRNA and has been shown to greatly increase expression of transgenes fused to the psaA promoter/5′UTR (Michelet et al 2011 ). Additionally, Ferreira-Camargo et al ( 2015 ) have reported that the inclusion of selenocystamine in the growth medium improves the accumulation of proteins containing disulfide bonds and could therefore also increase hGH yield.…”

The Potential for Microalgae as Bioreactors to Produce Pharmaceuticals

“…A new nuclear transformation method by microparticle bombardment was developed in Phaeodactylum tricornutum using PCR-amplified DNA fragments [ 83 ]. In addition, Ferreira-Camargo et al found that selenocystamine can improve accumulation of disulfide bonds-contained proteins in green algal chloroplasts [ 84 ].…”

Small molecule diselenide additives for in vitro oxidative protein folding

“…More recently, selenocystamine improved the accumulation of disulfide containing proteins in chloroplasts of eukaryotic green algae. 13 Selenium is a particularly effective electrophile and nucleophile in nucleophilic exchange reactions, 14,15 due to its greater polarizability in comparison to sulfur and the lower pK a for selenol side-chain of Sec compared to thiol of Cys (5.2 versus 8.5, respectively). 16 Owing to the rapid oxidation of selenols by atmospheric oxygen, catalytic amounts of diselenides can be used for protein refolding under aerobic conditions.…”

“…Acetonide 5 deprotection was achieved by TFA, providing diselenide 1 in quantitative crude yield. The product was purified via C 18 preparative reversed-phase (RP) HPLC and characterized by 1 H-, 13 C-and 77 Se-NMR and ESI-MS (see ESI †).…”