Optimization of Vasopressin Type 2 Receptor Expression in Escherichia coli BL21

Abstract

Background: Vasopressin type 2 receptor (V 2 R) is a G-protein-coupled receptor (GPCRs) located in nephrons mediating water reabsorption in kidneys. Vasopressin acts at these receptors to enhance water reabsorption in the kidneys while by acting on V1 receptors in blood vessels increases blood pressure. To study V 2 R better, it must be produced in high quantities. Objectives: The current study aimed to optimize expression of V 2 R in Escherichia coli. Materials and Methods: In this investigation, V 2 R gene was cloned in E. coli. The recombinant plasmid pEt15b/V 2 R (rpET-BL21) was transformed into competent E. coli strain BL21 (DE3) cells. Overnight culture of the transformed bacteria was induced by the addition of isopropyl-thio-β-D-galactoside (IPTG). The effects of different IPTG concentrations, temperatures and sampling times on the expression of V 2 R were examined. Samples were analyzed by SDS-PAGE. Results:The maximum amount of protein production was obtained by the addition of 0.75 mM IPTG at 37°C after three hours. The most important factor was harvesting time with P value < 0.0001. In statistical analysis of data, the normal plot shows the normality of distribution of the studentized data. Conclusions: Full factorial design experimental approach helped to identify the critical culture condition parameters in heterologous expression of V 2 R. It would allow studying the parameters affecting the function of this protein in future experiments.