Expression, Purification and Characterization of Functional Teduglutide Using GST Fusion System in Prokaryotic Cells.

Purpose: Teduglutide is the first and only FDA-approved drug for long-term treatment of short bowel syndrome (SBS). The current study aimed to present an approach for production of teduglutide using recombinant DNA technology. Methods: The coding gene for teduglutide was cloned into pGEX-2T vector, where coding sequence for factor Xa cleavage site was added between GST and teduglutide coding genes. The GST-teduglutide protein was overexpressed in E. coli BL21 (DE3) strain and affinity purified using glutathione sepharose affinity column. Results: On-column proteolytic activity of factor Xa followed by size exclusion chromatography resulted in the pure teduglutide. Circular dichroism (CD) spectropolarimetry showed that the produced teduglutide folds into mainly α-helical structure (>50%), as expected. In mass spectroscopy analysis, the fragments of teduglutide resulted by cyanogen bromide cleavage as well as those expected theoretically due to mass fragmentation were identified. The functionality of the produced peptide was evaluated by measuring its proliferative effect on Caco2 intestinal epithelial cells, and the results indicated that produced teduglutide induces cell proliferation by 19±0.30 and 33±7.82 % at 1.21 and 3.64 µM concentrations, respectively, compared to untreated cells. Conclusion: Teduglutide was successfully expressed and purified and its functionality and structural integrity were confirmed by in vitro experiments. We believe that the experimental-scale method presented in the current study can be useful for pilot-scale and also industrial-scale production of teduglutide.

Identification of Novel Mutations in Arabidopsis thaliana DOF 4.2 Coding Gene.

Purpose: DOF (DNA-binding with One Finger) proteins are plant-specific transcription factors which mediate numerous biological processes. The purpose of the current study is to report new naturally occurring mutations in the gene encoding for one of the members of DOF proteins named DOF 4.2. Methods: The expression of zinc finger domain of DOF 4.2 (DOF 4.2-ZF) was investigated by first synthesis of cDNA library using different parts of Arabidopsis thaliana plant. Then the coding sequence for zinc finger domain of DOF 4.2 protein was prepared using nested PCR experiment and cloned into pGEX-6P-1 expression vector. Finally, the prepared construct was used for protein expression. Furthermore, molecular dynamics (MD) simulation was carried out to predict DNA binding affinity of DOF 4.2-ZF using AMBER package. Results: For the first time a new variant of DOF 4.2-ZF protein with three mutations was detected. One of the mutations is silent while the other two mutations lead to amino acid replacement (S18G) as well as introduction of a stop codon ultimately resulting in a truncated protein production. In order to investigate whether the truncated form is able to recognize DNA binding motif, MD simulations were carried out and the results showed that the chance of binding of DOF 4.2-ZF protein to cognate DNA in its truncated form is very low. Conclusion: The findings demonstrated that the observed mutations adversely affect the DNA binding ability of the truncated form of DOF4.2 if it is expressed in the mutant variant of A. thaliana used in this study.

Expression, purification and molecular dynamics simulation of extracellular domain of glucagon-like peptide-2 receptor linked to teduglutide.

Teduglutide is the only drug approved for long-term treatment of short bowel syndrome (SBS). This drug exerts its pharmacological effects via binding to the GLP-2 receptors (ECD-GLP2R) located in intestinal tissue. The three dimensional (3D) structure of ECD-GLP2R hasn't been determined yet and hence its mode of interaction with agonists/antagonists is not clear. Therefore, it would be of great importance to develop a structural scaffold for investigation of ECD-GLP2R interactions with its binders. For this, the current study aimed to produce fusion protein of ECD-GLP2R-teduglutide. The ECD-GLP2R-teduglutide protein was expressed in bacterial expression system and purified using affinity and size exclusion chromatography techniques. Using circular dichroism the secondary structure content of purified protein was determined which was comparable to that of theoretical calculations. The low structural stability of purified protein (ΔG = 3.64 kJ.mol-1) was elucidated by monitoring its fluorescence emission at the presence of various concentrations of GdnHCl as a denaturant. Finally, a 3D model for ECD-GLP2R-teduglutide protein was generated and validated using molecular dynamics simulation whose information alongside the experimental studies can be useful for providing new insight into the mode of interaction of ECD-GLP2R with its specific ligands in order to design potent and specific GLP2R agonists.