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Investigating Stability and Solubility of Lysozyme Enzyme in Bacteria Cell through Protein Engineering and Computational Protein Design

Rad Kaftaroodi, Hassan | 2025

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  1. Type of Document: M.Sc. Thesis
  2. Language: Farsi
  3. Document No: 57894 (03)
  4. University: Sharif University of Technology
  5. Department: Chemistry
  6. Advisor(s): Kalhor, Hamid Reza
  7. Abstract:
  8. Due to its structural simplicity and functional importance, Hen egg white lysozyme (HEWL) is a widely studied model protein. However, its expression in E. coli often results in aggregation and the formation of inclusion bodies, limiting its utility for biotechnological applications. This study focuses on improving the solubility and stability of HEWL through a combination of experimental and computational approaches. In the first step, using error-prone PCR and the MEGAWHOP technique, libraries of potential HEWL variants were generated and screened via β-complementation for enhanced properties. To complement these efforts, computational tools such as ProteinMPNN and AlphaFold were employed to design novel HEWL sequences with reduced aggregation-prone regions. Molecular dynamics simulations were performed to validate the stability of these variants under various conditions, including oxidized and reduced states. The results demonstrated that the computationally optimized variants exhibited improved solubility and structural stability compared to the native HEWL, as confirmed by MD simulations. Redesigned HEWL retained secondary structure elements, displayed reduced aggregation-prone regions, and demonstrates improved solubility and thermal stability. Critical residues for enzymatic activity are preserved, ensuring functional integrity. These results suggest the new HEWL is a promising candidate for cytoplasmic expression in E. coli
  9. Keywords:
  10. Protein Engineering ; Protein Design ; Hen Egg White Lysozyme Enzyme ; Molecular Dynamic Simulation ; Membrane Protein Aggregation ; Protein Message Passing Neural Network (ProteinMPNN)Device ; MEGAWHOP Technique

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