title: Long-Term Stable and Tightly Controlled Expression of Recombinant Proteins in Antibiotics-Free Conditions.
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.
authors.affiliation: Synthetic Biology & Bioengineering Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Daejeon, Republic of Korea.
authors.forename: Soo-Jin
authors.initials: SJ
authors.lastname: Yeom
authors.affiliation: Synthetic Biology & Bioengineering Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Daejeon, Republic of Korea.|Biosystems & Bioengineering, University of Science & Technology (UST), Daejeon, Republic of Korea.
authors.forename: Dae-Hee
authors.initials: DH
authors.lastname: Lee
authors.affiliation: Synthetic Biology & Bioengineering Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Daejeon, Republic of Korea.|Department of Biological Sciences, Korea Advanced Institute of Science and Technology (KAIST), Daejeon, Korea.
authors.forename: Yu Jung
authors.initials: YJ
authors.lastname: Kim
authors.affiliation: Synthetic Biology & Bioengineering Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Daejeon, Republic of Korea.
authors.forename: Jeongmin
authors.initials: J
authors.lastname: Lee
authors.affiliation: Synthetic Biology & Bioengineering Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Daejeon, Republic of Korea.
authors.forename: Kil Koang
authors.initials: KK
authors.lastname: Kwon
authors.affiliation: Synthetic Biology & Bioengineering Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Daejeon, Republic of Korea.
authors.forename: Gui Hwan
authors.initials: GH
authors.lastname: Han
authors.affiliation: Synthetic Biology & Bioengineering Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Daejeon, Republic of Korea.
authors.forename: Haseong
authors.initials: H
authors.lastname: Kim
authors.affiliation: Department of Biological Sciences, Korea Advanced Institute of Science and Technology (KAIST), Daejeon, Korea.
authors.forename: Hak-Sung
authors.initials: HS
authors.lastname: Kim
authors.affiliation: Synthetic Biology & Bioengineering Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Daejeon, Republic of Korea.|Biosystems & Bioengineering, University of Science & Technology (UST), Daejeon, Republic of Korea.
authors.forename: Seung-Goo
authors.initials: SG
authors.lastname: Lee
mesh_terms: D000900:Anti-Bacterial Agents; D002338:Carotenoids / Q000096:biosynthesis; D002876:Chromosomes, Bacterial / Q000737:chemistry / Q000378:metabolism*; D003001:Cloning, Molecular / Q000379:methods*; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D005144:F Factor / Q000737:chemistry / Q000378:metabolism*; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D005810:Multigene Family; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics
chemical_list: D000900:Anti-Bacterial Agents; D011994:Recombinant Proteins; D049452:Green Fluorescent Proteins; D002338:Carotenoids