Li, Yiwen;
(2025)
Protein Engineering for Biotechnology.
Doctoral thesis (Ph.D), UCL (University College London).
Abstract
Rational design has emerged as a powerful strategy for protein engineering within biotechnology, specifically tailored to enhance the functional properties of proteins without the dependency on extensive library. The research initially focused on transketolase (TK), a critical enzyme in biocatalysis. By integrating non-natural amino acids, the efficiency of a novel reaction to form a pharmaceutical intermediate was enhanced. Employing a "push or pull" strategy, which utilised molecular dynamics simulations and substrate docking, the study achieved notable improvements in enzyme activity and thermal stability - enhancing the conversion rates by 17.7% and increasing thermal resilience by 6.7 °C. Expanding upon this foundation, the thesis explores the engineering of Adeno- Associated Virus (AAV) capsids, which are critical for gene therapy but face significant clinical and production challenges. A tailored engineering strategy was applied to the semi-conserved residues on surface loops, resulting in the creation of a series of capsid variants, notably RD14. This variant showcased a 21.2% enhancement in residual transduction efficacy after heat challenges, a 22.4-fold and 7.2-fold increase in muscle and heart infectivity, respectively, and a 4.70 nm increase in capsid diameter compared to AAV9. These results not only demonstrate the efficacy of the design approach but also provide valuable data to guide further iterative design efforts. Moreover, a comprehensive all-atom molecular dynamic simulation model of AAV capsid was developed for the first time, enabling detailed investigation of the mechanisms involved in AAV life cycles, which represents a significant step forward in understanding virus life cycles. In conclusion, this thesis demonstrates the efficiency and power of computation-guided rational design in protein engineering, establishing a new benchmark in the field and illustrating its vast potential for future biotechnological applications.
| Type: | Thesis (Doctoral) |
|---|---|
| Qualification: | Ph.D |
| Title: | Protein Engineering for Biotechnology |
| Language: | English |
| UCL classification: | UCL UCL > Provost and Vice Provost Offices > UCL BEAMS UCL > Provost and Vice Provost Offices > UCL BEAMS > Faculty of Engineering Science > Dept of Biochemical Engineering |
| URI: | https://discovery.ucl.ac.uk/id/eprint/10203466 |
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