| Summary: | Human papillomavirus (HPV) is a sexually transmitted virus and known precursor to
cervical cancer, the second most lethal cancer in females across the world. Two virus-like
particle (VLP) vaccines exist that provide immunity against the main serotypes of the
disease and are produced in Saccharomyces cerevisiae (S. cerevisiae) and baculovirus
infected insect cells. Pichia pastoris (P. pastoris) was chosen as an alternative expression
system for HPV VLP production based on its history as prolific heterologous protein
producer that circumvent many of the problems associated with aforementioned
expression systems. The strongly inducible AOX promoter allows three-phase
fermentations (1.3 L bioreactors) in which high cell densities (>100gCDW.L-1) are
obtained prior to induction with methanol. During the induction phase the dissolved
oxygen concentration may be used to control addition of methanol. It is also possible to
use predetermined methanol feed rates and to adjust the amount of additional oxygen
sparged to maintain a constant dissolved oxygen level. The effects of these control
strategies, different gene constructs and multiple gene integrations were quantified
through monomer-, VLP- and mRNA production levels.
Increased biomass concentrations in the 20% dissolved oxygen control strategy led to the
highest volumetric VLP concentration (68.53 mg.L-1). VLPs were located intracellularly
in both the cytoplasm and membranes of the yeast cells. Despite lower codon adaptation
of the h-L1 gene expressed in the X33[h-L1] strain it still had higher volumetric VLP
concentrations under 40% dissolved oxygen control than the X33[Syn-L1] and
X33[SA-L1] strain containing the SA-L1 and Syn-L1 genes. This was ascribed to the
possible presence of rare codons in the Syn-hL1 and SA-L1 genes and a lower A+T
content in the h-L1 gene. Multiple gene integrations of the h-L1 gene had a negative
effect on VLP production and this conclusion was supported by lower mRNA
concentrations indicating lower transcriptional efficiency. Increased methanol induction
efficiency in the DO control strategies was indicated by higher specific L1 monomer
levels. Decreased VLP to monomer ratios in the DO control strategies indicated that a
bottleneck 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
membranes within the yeast cells especially when efficient assembly to VLPs did not
occur. HPV16 L1 VLP concentrations obtained in P. pastoris in this study are
comparable to the study by Li et al., (2003), but much lower than expression levels
obtained in baculovirus infected insect cells. Based on the expression levels of HBsAg
VLPs obtained in P. pastoris, this system, with the necessary recommended optimisation,
has the capacity for increased HPV VLP production ability.
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