A new vector for heterologous gene expression in Escherichia coli with increased stability in the absence of antibiotic

Publication date: Available online 5 September 2018Source: PlasmidAuthor(s): Fara Amelia Primelles Eguia, Henrique Roman Ramos, Stefanie Kraschowetz, Daniel Omote, Celso Raul Romero Ramos, Paulo Lee Ho, Eneas Carvalho, Viviane Maimoni GonçalvesAbstractExpression 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 anti...
Source: Plasmid - Category: Biotechnology Source Type: research