Document Type : Research Paper

Authors

1 Molecular Medicine and Medical Biotechnology Department, Kurdistan University of Medical Sciences, Sanandaj, Iran.

2 Department of Plant Breeding, Faculty of Agriculture, Tarbiat Modares University, P.O. Box 14115-143, Tehran, IR Iran

3 Department of Biotechnology, Faculty of Agriculture, Tarbiat Modares University, Tehran, Iran

4 Department of Pharmacology and Toxicology Faculty of Pharmacy, Molecular Medicine Research Center, Hormozgan Health Institute, Hormozgan University of Medicinal sciences, Bandar Abbas, Iran.

10.30498/ijb.2022.308798.3175

Abstract

Background: Reteplase (recombinant plasminogen activator, r-PA) is a recombinant protein designed to imitate the endogenous tissue plasminogen activator and catalyze the plasmin production. It is known that the application of reteplase is limited by the complex production processes and protein’s stability challenges. Computational redesign of proteins has gained momentum in recent years, particularly as a powerful tool for improving protein stability and consequently its production efficiency. Hence, in the current study, we implemented computational approaches to improve r-PA conformational stability, which fairly correlates with protein's resistance to proteolysis.

Materials and Methods: Several web servers designed for mutation analysis were utilized to select appropriate mutations. Additionally, the experimentally reported mutation, R103S, converting wild type r-PA into non-cleavable form, was also employed. Firstly, mutant collection, consisting of 15 structures, was constructed based on the combinations of four designated mutations. Then, 3D structures were generated using MODELLER. Finally, 17 independent 20-ns molecular dynamics (MD) simulations were conducted and different analysis were performed like root-mean-square deviation (RMSD), root-mean-square fluctuations (RMSF), secondary structure analysis, number of hydrogen bonds, principal components analysis (PCA), eigenvector projection, and density analysis.

Results: Predicted mutations successfully compensated the more flexible conformation caused by R103S substitution, so, improved conformational stability was analyzed from MD simulations. In particular, R103S/A286I/G322I indicated the best results and remarkably enhanced the protein stability.

Conclusion: The conformational stability conferred by these mutations will probably lead to more protection of r-PA in protease-rich environments in various recombinant systems and potentially enhance its production and expression level.

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