Development of CYB5-fusion monitoring system for efficient periplasmic expression of multimeric proteins in Escherichia coli

Publication date: December 2016 Source:Protein Expression and Purification, Volume 128 Author(s): Dmitri Dormeshkin, Andrei Gilep, Gennady Sergeev, Sergey Usanov Despite all advances of heterologous expression of recombinant proteins in Escherichia coli, expression of multidomain disulphide-rich proteins faces some problems due to the absence of the possibility to monitor the process in real-time. Here we provide a CYB5-fusion system – cytochrome b 5 fusion system for periplasmic expression of multimeric proteins with the possibility of process monitoring. We validated this system by Fab and scFv antibody fragments expression in order to improve antibody-derived molecules characterization and to simplify their usage. The combination of redox dependent absorbance spectrum of red-colored cytochrome b 5 with its high value molar extinction coefficient allows us to monitor antibody fusion proteins localization, redox state and quantify them in reliable way in turbid solutions. Moreover, it was revealed that due to outstanding stability and solubility, cytochrome b 5 significantly enhances expression level of Fab/scFv antibody fragments in Escherichia coli periplasm. Graphical abstract
Source: Protein Expression and Purification - Category: Biochemistry Source Type: research