Design and preparation of genetic construct for the production of teduglutidE – GLP2R fusion protein in bacterial expression system

Abstract

Introduction: Short bowel syndrome (SBS), is a seriously disabling condition, which results from loss of substantial portions of intestine or its function, leading to inadequate absorption of nutrients and fluids. Teduglutide is the first drug which has been introduced for long-term treatment of SBS with beneficial effects approved in different clinical trials. This drug exerts its pharmacological effets via binding to the GLP-2 receptors (GLP2R) located in intestinal tissue. Aims:The current study aimed toproduce and purify GLP2R-Teduglutide fusion protein in order to future structural studies on mode of interactions of Teduglutide with GLP2R. Material and Methods: First, the synthesized DNA sequence of GLP2R-Teduglutide was cloned into pGEX-2T expression vector. Then the constructed vector was transformed into E.coli Bl21 and the protein of interest was expressed attached to glutathione S-transferase (GST) protein. Bacterial cells were disrupted and the whole protein content was applied onglutathione-Sepharose affinity column.After several washes GLP2R-Teduglutide fusion protein was cleaved off the column using thrombin proteolytic activity. The expression and purity of the produced protein was analyzed by western blotting and SDS-PAGE techniques. Results: The GLP2R-Teduglutide fused to GST was produced in bacterial expression system. The protein band at about 50 kDa on SDS-PAGE was attributed to GLP2R-Teduglutide-GST protein. The production of protein of interest was also confirmed by using western blotting technique. Finally, GLP2R-Teduglutide complex was cleaved off GSTusing thrombin activity. Conclusion: In the current work, GLP2R-Teduglutide fusion protein was successfully expressed and purified in a prokaryotic system. The produced fusion protein can be studied from the structural point of view using NMR and crystallographic methods in order to investigate the mode of interaction between GLP2R and Teduglutide.