Developing a biotechnological platform for the production of affinity reagents in aspergillus oryzae

Antibodies are important molecules produced by living organisms' immune systems in reaction to foreign particles or antigens. For many years, monoclonal antibodies (mAbs) have been employed for therapeutic, diagnostic, and imaging applications. However, it is well known that mAb production has been limited by recombinant technologies due to their large (150 kDa) and complex structure, which includes disulfide bonds and precise post-translational modification required for the functionality. The main difficulties encountered in producing mAbs on a large scale include solubility and folding issues in Escherichia coli (E. coli) expression systems and the requirement of expensive and time-consuming production methods for obtaining low levels of mAbs in mammalian expression systems. Single-domain heavy chain antibody (VHH), also referred to as a nanobody, is a distinct class of antibody that Hamers first identified in camelids in 1993 as a component of heavy-chain antibodies (HCAbs). Due to its size of 4 nm and 2.5 nm in diameter, VHH is approximately 13 kDa and free of the light (L)-chain. Despite the fact that VHH only has three complementarity-determining regions (CDRs), it ensures extremely specific antigen binding affinity when compared to typical mAbs, which have six CDRs to sustain binding to an antijen. Furthermore, the prolonged CDR3 loop of VHH expands the binding surface by forming a convex structure to bind the concave antigens with great specificity, allowing it to interact with antigens like enzymes that mAbs cannot. The hydrophobic amino acids in the VL domain of conventional mAbs were replaced with smaller hydrophilic amino acids to compensate for the lack of the VL domain in VHH. Therefore, VHH can easily penetrate tissues owing to its small size compared to mAbs. The immunogenicity of VHH is low as domains share high sequence identity with human VH, but it can be arranged if it is required as VHH is easily modified. Due to its small size, high specificity and affinity, resilience to heat and denaturing chemicals, low immunogenicity, and high tissue penetration capability, VHH possesses special qualities that make it suitable as an affinity reagent. High-yield heterologous expression of mAbs, which are utilized as affinity reagents in biotechnological applications, is hampered by issues with three-dimensional folding and solubility challenging, as well as the requirement for expensive production procedures. On the other hand, VHHs are unique antibody fragments that are smaller in size than mAbs and have considerable advantages over them. VHHs have begun to replace mAbs in various biotechnological applications, including diagnostic, therapeutic, and imaging technologies, due to their small size, low immunogenicity, and excellent solubility behaviour. Aspergillus oryzae (A. oryzae) is a filamentous fungus that has been preferred in fermentation technology and industrial-scale production of biotechnological products for many years, and has been given a Generally Safe (GRAS) status by the United States Food and Drug Administration (FDA). Compared to the taxonomically closest Aspergillus species, A. oryzae possesses more genes in its genome, especially in terms of genes encoding hydrolytic enzymes. A. oryzae's robust secretion system makes it an ideal platform for the large-scale and reliable production of affinity reagents. The purpose of this thesis is to construct a biotechnological platform using A. oryzae as a host, which has evolved to become pyrG auxotrophic, and to demonstrate that a pyrG auxotrophic A. oryzae expression platform can be used to produce affinity reagents. Additionally, it was desired to purify the anti-RNase A VHH protein, which is specific to ribonucleic acid A (RNase A), which was chosen as the affinity reagent, on a small scale and at the fermenter level. The wild type A. oryzae RIB40 host microorganism was initially transformed by a deletion vector constructed from the upstream and downstream regions of the pyrG gene's open reading frame, without pyrG gene. Thus, homologous recombination was used to eliminate the pyrG gene from the wild-type A. oryzae genome. The pyrG gene encodes the orotidine monophosphate decarboxylase enzyme, which plays a role in the pyrimidine pathway; therefore, a pyrG auxotroph colony develops resistance to 5-FOA (5-fluoroorotic acid) and requires uracil and uridine for survival. To confirm gene deletion, repetaed streaking method on selective culture medium and qPCR analysis were performed. The expression studies of the recombinant anti-RNase A VHH protein were carried out using the pyrG auxotroph A. oryzae bacterium, whose gene deletion was confirmed. The coding sequence of the anti-RNase A VHH protein, which was codon-optimized for A. oryzae, was synthesized under the amylase promoter and an 8xHis tag attached to the C-terminus of the protein. After the transformation was achieved by the protoplast-mediated transformation method and the confirmation studies of the colony expressing the recombinant anti-RNase A VHH protein were performed, small-scale expression was carried out. VHH was successfully produced and secreted in the soluble form at approximately 13 kDa. Following the successful implementation of small-scale expression, large-scale production was practiced in a 6 L fermenter. Recombinant anti-RNase A VHH, which was expressed in A. oryzae, was purified by immobilized metal affinity chromatography (IMAC) method utilizing the 8xHis-tag at the C-terminus. The production yield achieved from small-scale expression was calculated to be 44 mg/mL, and the yield from expression in the fermenter was calculated to be 1.4 g/L. Gel filtration chromatography was conducted for further purification, and it was demonstrated that the expressed recombinant anti-RNase A VHH protein is monomeric. Total produced protein and expressed recombinant protein concentration were analyzed densitometrically and determined by the Bradford assay using bovine serum albumin (BSA) as a standard. Pull-down, gel filtration, and surface plasmon resonance (SPR) analyses were completed to ascertain the spesific interaction of anti-RNase A VHH with its target antigen, RNase A. Gel filtration chromatography assay revealed that the expressed anti-RNAse A VHH possessed the correct three-dimensional folding structure and could bind with its target antigen, while pull-down assay confirmed the existence of a spesific interaction between anti-RNase A VHH and RNase A. The binding affinity of anti-RNase A VHH expressed in A. oryzae against RNase A was determined to be 1.9 nM in the SPR analysis. According to the results, the anti-RNase A VHH affinity reagent was successfully purified on a large scale with a high binding affinity, resulting in the correct three-dimensional structure and monomeric behaviour on the the established pyrG auxotroph A. oryzae platform. From this perspective, the conclusions reached within the scope of the thesis prove that the pyrG auxotroph A. oryzae expression system, thanks to its strong secretion ability, is an ideal platform for the cost-effective production of VHH affinity reagents, which are required in various biotechnological applications and research studies, in high volume and with high binding affinity to the target antigen.