Selenocystamine improves protein accumulation in chloroplasts of eukaryotic green algae
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- 作者：Livia S Ferreira-Camargo ; Miller Tran ; Joris Beld ; Michael D Burkart…
- 关键词：Chlamydomonas reinhardtii ; Selenocystamine ; Chloroplast ; Protein expression ; Recombinant protein
- 刊名：AMB Express
- 出版年：2015
- 出版时间：December 2015
- 年：2015
- 卷：5
- 期：1
- 全文大小：1,868 KB
- 刊物类别：Chemistry and Materials Science
- 刊物主题：Chemistry
Biotechnology
Microbiology
Microbial Genetics and Genomics - 出版者：Springer Berlin / Heidelberg
- ISSN：2191-0855
文摘
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.