Publication Date
2023
Document Type
Dissertation/Thesis
First Advisor
Horn, James R.
Degree Name
Ph.D. (Doctor of Philosophy)
Legacy Department
Department of Chemistry and Biochemistry
Abstract
The biophysical properties of the VHH domain from heavy-chain-only antibodies obtained from the Camelidae family has expanded opportunities for novel antibody applications. The small, single domain nature of VHH domains makes them attractive modules for fusion proteins. Here, two VHH fusion systems were evaluated, RNase A-anti-RNase A VHH and GFPMut3aR80Q-anti-GFP VHH, to investigate how antigen-VHH fusions may enhance protein thermostability. The introduction of various length flexible linkers was hypothesized to play a role in stability, as well as influence the oligomeric state. Bovine Pancreatic Ribonuclease A, or RNase A, is an enzyme belonging to the ribonuclease A superfamily, which is found in the pancreas of cows (Bos taurus). RNase A is a 13.7 kDa endoribonuclease that cleaves RNA molecules internally at P–O5′ bonds. Due to its specific catalytic activity, it has been tested for use in medication applications, where it has exhibited anti-tumor activity, and anti-viral activity against RNA viruses, such as hepatitis C and COVID-19. Here, an anti-RNase A VHH was used to create fusion proteins with RNase A using different sized linkers composed of GGGGS repeats. In addition, affinity-matured (Mat) VHH variants, which possess 100-fold higher affinity for RNase A, were examined. The 0x fusion behaved both as a monomer and a dimer in solution, while the 1x GGGGS linker fusion behaved as a dimer. Differential Scanning Fluorimetry (DSF) revealed that RNase A-anti-RNase A VHH 0x tandem monomer showed the largest increase in melting temperature, Tm, with an increase of ~25 ℃ over that observed for RNase A. The 1x fusion variant and the affinity-matured variants, using DSF and circular dichroism (CD) showed an increase in Tm of up to ~13 °C over RNase A. The affinity-matured RNase A-anti-RNase A VHH 0x trimer or larger species had a Tm decrease of ~3 °C over wildtype, observing an affinity-stability trade-off. Chemical stability studies suggested the 0x monomer and dimer fusion variants possessed higher mid-points of chemical unfolding but did not appear to be fully reversible under the conditions tested. Finally, the enzyme activity of the fusion variants was evaluated using a fluorescent assay, which revealed that all were active at 25 °C and 45 °C, with the 0x and 1x tandem variants exhibiting a 71- to 136-fold higher activity over RNase A, respectively. Green fluorescent protein (GFP) and its variants are widely used as molecular probes and reporters in biological imaging and biosensing applications. These applications can involve exposing GFP to different environments, including elevated temperatures in certain experimental setups. An anti-GFP VHH was used to create fusion variants with GFP containing different GGGGS linkers of 1x, 2x, and 3x. In addition, a 3x variant containing an additional disulfide bond within the VHH domain was generated. All variants were found to exist as monomeric species in solution. The excitation and emission of the GFP-anti-GFP VHH variants closely matched that of the GFPMut3aR80Q, 501 nm and 511 nm, respectively, suggesting that the fluorescence profile of GFP did not appreciably change when bound to VHH. The melting points were determined using fluorescence temperature melts, which revealed that the GFP-anti-GFP VHH 2x, 3x, and 3x-DS variants, displayed an increase in Tm over wildtype by ~7 to 13 °C. CD-based melting experiments revealed that a ~10 to 17 °C increase in Tm was observed for the GFP-anti-GFP VHH 2x and 3x tandems. Generation of a synthetic VHH library can be used to discover novel antibodies against antigens of interest. Consequently, a phage display library was designed using doped degenerate oligos, as an alternative to phosphoramidite-generated oligos, which are significantly more expensive. The amino acid incorporation aimed to match those commonly found in existing VHH domain structures. The anti-GFP VHH gene was introduced into a phagemid vector, upstream of gene III, using overlap extension mutagenesis. Afterwards, ssDNA was generated, and oligonucleotide-directed mutagenesis was conducted to introduce modifications to complementarity determining regions (CDRs) 1, 2, and 3.
Recommended Citation
Patel, Niyati, "Characterizing VHH Domains as Stabilization and Oligomeric Control Modules" (2023). Graduate Research Theses & Dissertations. 7841.
https://huskiecommons.lib.niu.edu/allgraduate-thesesdissertations/7841
Extent
217 pages
Language
en
Publisher
Northern Illinois University
Rights Statement
In Copyright
Rights Statement 2
NIU theses are protected by copyright. They may be viewed from Huskie Commons for any purpose, but reproduction or distribution in any format is prohibited without the written permission of the authors.
Media Type
Text
