Selenocystamine improves protein accumulation in chloroplasts of eukaryotic green algae
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.
- Research Organization:
- Univ. of California, San Diego, CA (United States)
- Sponsoring Organization:
- USDOE Office of Energy Efficiency and Renewable Energy (EERE); National Science Foundation (NSF); Chemical, Bioengineering, Environmental, and Transport Systems (CBET); California Energy Commission, California Initiative for Large Molecule Sustainable Fuels (CILMSF); National Institutes of Health (NIH)
- Grant/Contract Number:
- EE0003373; 1160184; 500-10-039; R01GM094924; R01GM095970
- OSTI ID:
- 1503061
- Alternate ID(s):
- OSTI ID: 1627034
- Journal Information:
- AMB Express, Journal Name: AMB Express Vol. 5 Journal Issue: 1; ISSN 2191-0855
- Publisher:
- Springer Science + Business MediaCopyright Statement
- Country of Publication:
- Germany
- Language:
- English
Web of Science
New tools for chloroplast genetic engineering allow the synthesis of human growth hormone in the green alga Chlamydomonas reinhardtii
|
journal | February 2016 |
The Potential for Microalgae as Bioreactors to Produce Pharmaceuticals
|
journal | June 2016 |
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