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Abstract Expression vectors for industrial production should be stable and allow tight control of protein synthesis. This is necessary to ensure plasmid transmission to daughter cells in order to achieve a stable population capable of synthesizing high amounts of the target protein. A high-copy-number plasmid, pAE, was previously used for laboratory-scale production of recombinant human granulocyte colony-stimulating factor (rhG-CSF) and the Schistosoma mansoni fatty acid binding protein (rSm14), but it was unstable for large-scale production. Therefore, here we evaluated a new expression vector derived from pAE, pAR-KanI, which combines two plasmid replication strategies: a high-copy plasmid pUC origin of replication as pAE, and a par locus sequence derived from pSC101, which is typical of low copy plasmids, for rhG-CSF and rSm14 production in Escherichia coli. Clones bearing these constructs were cultivated in two complex media (2YT and auto-induction) and both yielded higher-than-95% resistant colonies, before and after induction, either with or without antibiotics. In 2YT medium, we obtained 244 μg/mL of rSm14, 181 μg/mL and 392 μg/mL for rhG-CSF, with and without glucose, respectively. In auto-induction medium without antibiotics, 147 μg/mL of rSm14 and 162 μg/mL of rhG-CSF were obtained. The new vector presented high stability for the production of both recombinant proteins in complex media in Escherichia coli , even in the absence of antibiotics, making the pAR-KanI a promising vector for industrial production of recombinant proteins. Highlights • The effect of a par locus insertion into a high copy number plasmid on the stability was evaluated. • The novel expression vector pAR-KanI presented high stability in E. coli c ultivated in complex media without antibiotics. • pAR-KanI was successfully employed for production of two important molecules: rhG-CSF and rSm14. • The absence of antibiotics is important to reduce the costs of the recombinant protein production process. [ABSTRACT FROM AUTHOR]