title: 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.
authors.affiliation: Division of Biological Sciences, UC San Diego, 9500 Gilman Drive, Bonner Hall, La Jolla, CA, 92093-0368, USA, liviaseno@hotmail.com.
authors.forename: Livia S
authors.initials: LS
authors.lastname: Ferreira-Camargo
authors.forename: Miller
authors.initials: M
authors.lastname: Tran
authors.forename: Joris
authors.initials: J
authors.lastname: Beld
authors.forename: Michael D
authors.initials: MD
authors.lastname: Burkart
authors.forename: Stephen P
authors.initials: SP
authors.lastname: Mayfield