Abstract
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.
Highlights
Plasmid-based protein production has been an essential tool for several decades in the field of biotechnology because of the ease of construction and ability to transfer the same plasmid into multiple strains [1,2]
The pRMT system was used successfully for in vivo cloning, the expression of target genes still requires the selective antibiotic pressure to maintain the plasmid in cells
We attempted the integration of pRMT into the E. coli JM109 chromosome at the location of the bglA gene, which resulted in a significant decrease of protein expression [19]
Summary
Plasmid-based protein production has been an essential tool for several decades in the field of biotechnology because of the ease of construction and ability to transfer the same plasmid into multiple strains [1,2]. This approach allows for the overexpression of genes of interest, generating large amounts of recombinant proteins [3]. An important limitation in the production of recombinant proteins using common plasmids in Escherichia coli is the requirement for the selective pressure of an antibiotic to be stably maintained in cells, PLOS ONE | DOI:10.1371/journal.pone.0166890. An important limitation in the production of recombinant proteins using common plasmids in Escherichia coli is the requirement for the selective pressure of an antibiotic to be stably maintained in cells, PLOS ONE | DOI:10.1371/journal.pone.0166890 December 1, 2016
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