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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 ofmore » 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.« less

Authors:
; ; ; ;
Publication Date:
Research Org.:
Univ. of California, San Diego, CA (United States)
Sponsoring Org.:
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)
OSTI Identifier:
1503061
Alternate Identifier(s):
OSTI ID: 1627034
Grant/Contract Number:  
EE0003373; 1160184; 500-10-039; R01GM094924; R01GM095970
Resource Type:
Published Article
Journal Name:
AMB Express
Additional Journal Information:
Journal Name: AMB Express Journal Volume: 5 Journal Issue: 1; Journal ID: ISSN 2191-0855
Publisher:
Springer Science + Business Media
Country of Publication:
Germany
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; Biotechnology & Applied Microbiology

Citation Formats

Ferreira-Camargo, Livia S., Tran, Miller, Beld, Joris, Burkart, Michael D., and Mayfield, Stephen P. Selenocystamine improves protein accumulation in chloroplasts of eukaryotic green algae. Germany: N. p., 2015. Web. doi:10.1186/s13568-015-0126-3.
Ferreira-Camargo, Livia S., Tran, Miller, Beld, Joris, Burkart, Michael D., & Mayfield, Stephen P. Selenocystamine improves protein accumulation in chloroplasts of eukaryotic green algae. Germany. doi:10.1186/s13568-015-0126-3.
Ferreira-Camargo, Livia S., Tran, Miller, Beld, Joris, Burkart, Michael D., and Mayfield, Stephen P. Fri . "Selenocystamine improves protein accumulation in chloroplasts of eukaryotic green algae". Germany. doi:10.1186/s13568-015-0126-3.
@article{osti_1503061,
title = {Selenocystamine improves protein accumulation in chloroplasts of eukaryotic green algae},
author = {Ferreira-Camargo, Livia S. and Tran, Miller and Beld, Joris and Burkart, Michael D. and Mayfield, Stephen P.},
abstractNote = {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.},
doi = {10.1186/s13568-015-0126-3},
journal = {AMB Express},
number = 1,
volume = 5,
place = {Germany},
year = {2015},
month = {7}
}

Journal Article:
Free Publicly Available Full Text
Publisher's Version of Record
DOI: 10.1186/s13568-015-0126-3

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Cited by: 3 works
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