Development of Pichia pastoris as a production system for HPV16 L1 virus-like particles as component to a subunit vaccine
Year: 2007
Type: dissertation
Abstract: Human papillomavirus (HPV) is a sexually transmitted virus and known precursor to \ncervical cancer, the second most lethal cancer in females across the world. Two virus-like \nparticle (VLP) vaccines exist that provide immunity against the main serotypes of the \ndisease and are produced in Saccharomyces cerevisiae (S. cerevisiae) and baculovirus \ninfected insect cells. Pichia pastoris (P. pastoris) was chosen as an alternative expression \nsystem for HPV VLP production based on its history as prolific heterologous protein \nproducer that circumvent many of the problems associated with aforementioned \nexpression systems. The strongly inducible AOX promoter allows three-phase \nfermentations (1.3 L bioreactors) in which high cell densities (>100gCDW.L-1) are \nobtained prior to induction with methanol. During the induction phase the dissolved \noxygen concentration may be used to control addition of methanol. It is also possible to \nuse predetermined methanol feed rates and to adjust the amount of additional oxygen \nsparged to maintain a constant dissolved oxygen level. The effects of these control \nstrategies, different gene constructs and multiple gene integrations were quantified \nthrough monomer-, VLP- and mRNA production levels. \nIncreased biomass concentrations in the 20% dissolved oxygen control strategy led to the \nhighest volumetric VLP concentration (68.53 mg.L-1). VLPs were located intracellularly \nin both the cytoplasm and membranes of the yeast cells. Despite lower codon adaptation \nof the h-L1 gene expressed in the X33[h-L1] strain it still had higher volumetric VLP \nconcentrations under 40% dissolved oxygen control than the X33[Syn-L1] and \nX33[SA-L1] strain containing the SA-L1 and Syn-L1 genes. This was ascribed to the \npossible presence of rare codons in the Syn-hL1 and SA-L1 genes and a lower A+T \ncontent in the h-L1 gene. Multiple gene integrations of the h-L1 gene had a negative \neffect on VLP production and this conclusion was supported by lower mRNA \nconcentrations indicating lower transcriptional efficiency. Increased methanol induction \nefficiency in the DO control strategies was indicated by higher specific L1 monomer \nlevels. Decreased VLP to monomer ratios in the DO control strategies indicated that a \nbottleneck existed in the assembly process due to increased L1 monomer concentrations. Due to the hydrophobic region on the L1 protein, these proteins associated with the \nmembranes within the yeast cells especially when efficient assembly to VLPs did not \noccur. HPV16 L1 VLP concentrations obtained in P. pastoris in this study are \ncomparable to the study by Li et al., (2003), but much lower than expression levels \nobtained in baculovirus infected insect cells. Based on the expression levels of HBsAg \nVLPs obtained in P. pastoris, this system, with the necessary recommended optimisation, \nhas the capacity for increased HPV VLP production ability.
Author: Lara Kotze
Language: English
Subfield: Molecular Biology
Domain: Life Sciences
Open Access status: green
OpenAlex ID: W2156604953