Doctoral Degrees (Microbiology)

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Browsing Doctoral Degrees (Microbiology) by Author "Bredell, Wilhelmina Jacoba"

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    Investigation of methylotrophic yeasts expression systems for the production of self-assembling proteins with biological applications
    (Stellenbosch : Stellenbosch University, 2018-12) Bredell, Wilhelmina Jacoba; Van Zyl, Willem Heber; Gorgens, Johann F.; Stellenbosch University. Faculty of Science. Dept. of Microbiology.
    ENGLISH ABSTRACT: Functional biological proteins are gaining interest with potential application in medicine and biotechnology. The methylotrophic yeasts, especially Pichia pastoris and Hansenula polymorpha, have been successfully used for the production of heterologous proteins with high industrial and medicinal importance. The aim of this study was to explore the production of self-assembly proteins in methylotrophic yeasts. The specific proteins investigated were the Human Papilloma virus (HPV) and Rotavirus (RV) proteins that self-assemble into virus-like particles (VLPs), and the α-chain proteins that assembles into procollagen. Cervical cancer is ranked the fourth most common cancer in women worldwide. Human Papilloma virus types 16 and 18 account for 70% of all cervical cancers worldwide. Despite the availability of two commercial prophylactic vaccines, it is unaffordable for most women in developing countries. We compared the optimized expression of monomers of the unique HPV type 16 L1/L2 chimeric protein (SAF) in two yeast strains of P. pastoris, KM71 (MutS) and GS115 (Mut+), together with H. polymorpha NCYC 495, to determine the preferred host in bioreactors. SAF was uniquely created by replacing the h4 helix of the HPV type 16 capsid L1 protein with a 13-amino acid peptide from the L2 protein. Two different methanol-feeding strategies in fed-batch cultures of P. pastoris MutS were evaluated: a predetermined feed rate versus feeding based on the oxygen consumption by maintaining constant dissolved oxygen levels (DO stat). All cultures showed a significant increase in biomass concentrations when methanol was fed using the DO stat method. In P. pastoris, the SAF concentrations were higher in the MutS strains than in the Mut+ strains. Our results showed the maximum concentration SAF expressed in both yeasts reported to date with H. polymorpha the best producer of all the yeasts evaluated. Further, accurate quantification of SAF was obtained through direct comparison with known concentrations of purified HPV16 L1. Quantification is usually inferred from ELISA results using standards not always compatible to the L1 monomer. Previous research in insect cells showed that the HPV16 L1/L2 chimeric protein self-assembled in capsomeres or capsomer aggregates during expression in insect cells. With the human codon-optimized chimeric gene, occational T=7 VLPs were visible in the insect cells. Similarly, when SAF was expressed in both methylotrophic yeasts, the majority of heterologous protein was observed as capsomeres (10 nm in diameter) with the occasional T=1 VLPs (25-30 nm in diameter) assembled. This is the first report showing the formation of T=1 VLPs in H. polymorhpa. SAF proteins displaying L2 epitopes offer simultaneously high titres of L1 specific neutralizing antibodies, as well as cross-neutralizing antibodies against L2. Gastroenteritis is one of the leading causes of deaths in children under the age of five years worldwide and caused by RV. We expressed a RV VP6 protein, derived from a prevalent South African RV strain (G9P), intracellularly in Escherichia coli, P. pastoris and H. polymorpha. Despite producing the lowest biomass levels of all the expression systems in shake flasks, the highest VP6 concentration was obtained with E. coli. In the controlled environment of bioreactors, all three expression systems attained higher cell densities and increased growth-associated VP6 production, in comparison to shakeflasks. Unlike in shake flask expressions, H. polymorpha outperformed both P. pastoris and E. coli during bioreactor cultivation. In contrast to yeast expressions, bacterial expressed VP6 protein was found to be insoluble upon analysis. This is the first report of VP6 expressed in methylotrophic yeast and holds the promise for the inexpensive production of VP6 as a possible vaccine candidate, booster dose or drug delivery mechanism. Collagen is the main structural protein of various animal connective tissues and also has the natural ability to self-assemble; therefor, it poses similar expression challenges than VLPs. Treatment of burn victims can benefit from combining a collagen α-chain with an antimicrobial peptide (AMP) in wound dressings. The collagen can aid in wound healing and the AMP in fighting infectious agents present. This collagen-AMP fusion protein was extracellularly produced by H. polymorpha. Presence of a correctly-sized single band on tricine-SDS PAGE gels revealed the successful expression of the putative fusion protein. Proteomic analysis of this protein species only identified part of this fusion. This is the first evidence of the possible successful expression of a recombinant collagen-AMP fusion protein in yeast. This study suggests that H. polymorpha is the preferred host, among the host cells tested, for the production of self-assembly proteins, such as the protein-components of virus-like particles and structural fusion proteins, such as collagen-AMP.