Long-Term Stable and Tightly Controlled Expression of Recombinant Proteins in Antibiotics-Free Conditions

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Plasmid-based gene expression is a fundamental tool in the field of biotechnology. However, overexpression of genes of interest with multi-copy plasmids often causes detrimental effects on host cells. To overcome this problem, chromosomal integration of target genes has been used for decades; however, insufficient protein expression occurred with this method. In this study, we developed a novel cloning and expression system named the chromosomal vector (ChroV) system, that has features of stable and high expression of target genes on the F' plasmid in the Escherichia coli JM109(DE3) strain. We used an RMT cluster (KCTC 11994BP) containing a silent cat gene from a previous study to clone a gene into the F' plasmid. The ChroV system was applied to clone two model targets, GFPuv and carotenoids gene clusters (4 kb), and successfully used to prove the inducible tightly regulated protein expression in the F' plasmid. In addition, we verified that the expression of heterologous genes in ChroV system maintained stably in the absence of antibiotics for 1 week, indicating ChroV system is applicable to antibiotics-free production of valuable proteins. This protocol can be widely applied to recombinant protein expression for antibiotics-free, stable, and genome-based expression, providing a new platform for recombinant protein synthesis in E. coli. Overall, our approach can be widely used for the economical and industrial production of proteins in E. coli.
Publisher
PUBLIC LIBRARY SCIENCE
Issue Date
2016-12
Language
English
Article Type
Article
Keywords

ESCHERICHIA-COLI; CHROMOSOMAL INTEGRATION; PLASMID SELECTION; GENE-EXPRESSION; EFFICIENT; DNA; REPLICATION; EXCISION; VECTORS; SYSTEMS

Citation

PLOS ONE, v.11, no.12

ISSN
1932-6203
DOI
10.1371/journal.pone.0166890
URI
http://hdl.handle.net/10203/216131
Appears in Collection
BS-Journal Papers(저널논문)
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