title: Advancing the use and development of transient expression in plants
Creators: name: Venturi, Micol
Publisher: name: Griffith University
Year: 2024
Subjects: subject: transient expression
subject: VLPs purification
subject: PHB beads
subject: plasmid copy number

description: The use of plants to produce recombinant products has advanced tremendously over the past two decades. It is now capable of competing with traditional expression systems for market share, with advantages in terms of low upstream cost, ease of scale-up and safety. The major plant-based platforms for heterologous protein production are cell suspension culture, transient expression and transgenic plants. Transient expression finds application in many fields, for instance, synthetic biology and metabolic engineering, in addition to biopharmaceutical manufacturing, and its outstanding feature is the rapidity with which it is possible to accumulate recombinant protein in the leaf tissue. This is achieved thanks to the infection mechanism of Agrobacterium tumefaciens combined with agroinfiltration in mature biomass that allows the production of recombinant proteins in less than seven days. This study highlights the speed of transient expression, exemplifying its utility in various technological applications. Virus-like particles (VLPs), non-infectious nanoparticles derived from viruses lacking genetic material, have garnered significant interest in biotechnology and nanotechnology. Their unique attributes, including self-assembly, stability, heterogeneity, immunogenicity, and biocompatibility, render them versatile candidates for various applications. To harness the full potential of VLPs, a comprehensive understanding of the mechanisms governing their assembly, disassembly, and intricate interactions with host systems is imperative. The study of VLPs as structural mimics of viruses is a growing trend in structural virology, yet it faces a critical bottleneck in the preparation of high quality samples. A fundamental aspect of this work involves the development of a simple and high-performing protocol for VLP purification. The purification process is essential for obtaining high-quality VLP samples, facilitating in-depth studies of their structural and functional attributes through, for instance, cryogenic electron microscopy. This thesis also explores the flexibility of transient expression in the context of a metabolic engineering endeavour focused on producing polyhydroxybutyrate (PHB), a biopolymer with significant industrial and medical relevance. Breaking new ground, PHB synthesis was achieved in Nicotiana benthamiana through transient expression, marking the first instance of such accomplishment. The investigation involved testing four distinct constructs identifying the most efficient configuration for high PHB accumulation in the cytosol. This achievement lays the groundwork for future studies and opens avenues for developing a plant PHB production platform. Moreover, this work investigates a frequently neglected aspect of binary vectors-their replication system. Building on anecdotal evidence suggesting a potential link between plasmid copy number (PCN) and protein expression levels, experimental evidence is presented to substantiate this link. The research explores the correlation between PCN and protein expression in plants by employing RK2 copy-up mutants as the primary approach and subsequently examining the replication machinery from existing binary vectors as an alternative strategy; the study offers compelling support for the hypothesised relationship. This work provides valuable insights for developing a more efficient transient expression binary vector. Overall, this thesis advances the use and development of transient expression in plants for the benefit of a broad range of bioscience research.