Date of Award

2024

Document Type

Dissertation

Degree Name

Philosophy (Ph.D)

Department

Pharmaceutical Sciences

First Advisor

Woon-Kai Low

Abstract

Adeno-associated virus (AAV) is a widely used vector for gene therapy. However, few studies have focused on producing AAV-based virus-like particles (VLP) in E. coli. This project aimed to express VP3 of AAV2 or co-express VP3 of AAV2 with other proteins, such as E. coli chaperones and/or assembly-activating protein (AAP) in E. coli under different expression conditions and explore the potential to produce VP3-only VLP of AAV2 in situ in E. coli. The constructed plasmid pET15ΔHis-VP3 was used to express VP3 under different conditions. The results demonstrated that most expressed VP3 was in inclusion bodies in all tested conditions. Although low level of soluble VP3 could be produced in E. coli after optimization, those proteins might be present as monomers or aggregates. To promote the assembly of VP3, the constructed plasmid pET15ΔHis-VP3-AAP was used to co-express VP3 and AAP in E. coli. Assembled VP3-only VLP could be detected with dot blot assays and transmission electron microscopy after co-expression at 30 °C. However, the overall production level of VP3 after co-expression was lower than that after expression of VP3 alone. Therefore, we tried co-expression VP3 with E. coli chaperones (trigger factor, Hsp70 and chaperonin) instead of AAP. It showed that those chaperones were able to facilitate soluble expression of VP3 and prevent its aggregation, but only Hsp70 and chaperonin had small effects on promoting VP3-only VLP assembly. Triple co-expression of VP3, AAP and chaperones using E. coli (BL21 (DE3)) demonstrated that chaperonin could increase the production of VP3-only VLP when triple co-expressing VP3, AAP and chaperonin. Purification of VP3-only VLP of AAV2 was also attempted. Although partial purification could be achieved with polyethylenimine (PEI) precipitation followed by size exclusion chromatography with Sephacryl S-300 HR, further purification was hampered by the low yields. In conclusion, our results demonstrated that the in situ production of VP3-only VLP of AAV2 in E. coli was possible. However, further optimizations of expression conditions as well as purification conditions may be necessary to achieve a high yield of produced VLP.

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