Engineering proteins for covalent reactivity

Thesis

NeissLock technology employs a self-processing module derived from Neisseria meningitidis to drive calcium-inducible, protein:protein covalent conjugation using only the twenty naturally occurring amino acids. The original iteration of the NeissLock technology relied on existing protein-protein interactions and therefore could not be applied towards covalent modification of proteins without an endogenous ligand. In this thesis, I present the NanoBondy- an engineered nanobody capable of inducible conjugation to a specific protein target at the unmodified cell surface. Beginning from nanobodies to CD45 and to mouse IgG, I designed covalently-reactive NanoBondies. For the lead anti-CD45 NanoBondy candidate, I used computational modelling to predict the NanoBondy/CD45 binding interface. I evaluated various disulfide clamp sites and linker lengths to improve the overall efficiency of the NanoBondy reaction. I also evaluated the NanoBondy reaction tolerance to various buffers and reaction temperatures. In collaboration with University College London, we conducted crosslinking mass spectrometry to characterize the isopeptide crosslinks between the NanoBondy and CD45. I demonstrated NanoBondy conjugation to the surface of natural killer cells and CD8+ T cells. To apply the NanoBondy technology, I constructed DuoBondy constructs, consisting of a NanoBondy with an N-terminal effector protein. DuoBondy constructs maintain covalent reactivity to CD45 in recombinant protein assays. I evaluated the functional effect of DuoBondy binding in CD8+ T cell activation assays. The described NanoBondy technology offers a new means of covalent cell surface modification, as well as a novel protein-protein coupling tool with potential applications in protein conjugation, SPR and structural determination.

Authors: Vankayala, LR

• DOI: 10.5287/ora-xmvxbbarx
• Type of award: DPhil
• Level of award: Doctoral
• Awarding institution: University of Oxford
• Language: English
• Keywords: protein-protein conjugation; covalent; NeissLock; cell decoration; conjugation; SpyTag; nanobody
• Subjects: Protein engineering; Biochemistry