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Engineering protein and cell surface display platforms

Lopez Morales, Joanan. Engineering protein and cell surface display platforms. 2023, Doctoral Thesis, University of Basel, Faculty of Science.

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Abstract

The biological engineering of systems and proteins often requires exploring a vast collection of variants derived from a common protein parent template. Recently, machine learning has pushed the field of protein engineering by demanding high-quality large-scale data from experiments to create applicable models. This improvement in computational methods stimulates a need for better experimental tools for protein engineering in order to generate large scale training data, and efficiently isolate novel sequences with enhanced properties. In vitro display technologies are crucial to protein engineering campaigns, as they provide means to correlate the genetic information of a variant sequence with its resulting phenotypic properties. Such correlation, called the genotype-phenotype linkage, is necessary for high-throughput screening of combinatorial variant libraries. Yeast surface display is one of the most extensively used in vitro cell display systems due to its advantages, such as folding machinery, post-translational modifications, simultaneous detection of full-length expression, and binding. Displaying proteins on the surface of Saccharomyces cerevisiae yeast cells has enabled the development of powerful techniques to study protein-protein interactions and to evolve new functions in proteins of interest. Latest advances and improvements to the platform have focused on constructing new plasmids with different epitope tags or conjugation handles, broadening the plasmid repository. However, there is an urgent need for expanding the use of the yeast display outside its original objective, set back in 1997. Little work has been done to make yeast display compatible with other methods and setups. This thesis aimed to engineer new systems and proteins with enhanced properties using yeast surface display as a core technology platform. In the following chapters, we describe the design and implementation of three novel approaches that extend the capabilities of the yeast display platform and the range of its applications in biochemical and clinical settings. Chapter 1 presents a general introduction to the concepts and theoretical context of biomolecular interactions, focusing on: models to obtain binding constants under different conditions; protein display platforms with an in-depth description of yeast surface display; a state-of-the-art review of selected non-antibody scaffolds; and biophysical methodologies employed to characterize protein properties and their interactions. Chapter 2 describes: the development of a serological immunoassay to detect the presence of anti-SARS-CoV-2 immunoglobulins in human sera; the multiplexed characterization of binding profiles to different variants of concern; and the quantification of immune response profiles after vaccination or recovery from COVID-19. Here, the yeast immunoassay correctly detected antibodies against the WT (Wuhan), Delta, and Omicron SARS-CoV-2 variants. We performed multiplexed serology testing of anti-SARS-CoV-2 IgG on 35 serum samples of convalescent, vaccinated, and negative individuals, and identified binding to specific RBD variants with 100% specificity and 92% sensitivity. We employed the yeast assay to verify the immune escape of the Omicron variant. We observed significant differences between the RBD WT and Delta to the Omicron variant IgG responses (20 to 100-fold). Finally, we were able to discern the overall effect of vaccination schemes on IgG titer distributions using the yeast immunoassay. Chapter 3 presents: the development of a yeast surface display system with the capacity to titrate the displayed protein density; the characterization of protein display profiles using model molecules; the regulation of three relevant biological activities; and the enhancement of high-throughput screening methods by coupling the yeast titratable system. The platform controls the density of displayed proteins per cell by a titratable gene circuit in the cells. The synthetic controller gives negative feedback to expressed protein fusions in a titrated manner, dependent on tetracycline. We employed the yeast titratable display platform to regulate the expression and display of a homodimeric enzyme, an adhesion receptor, and a non-antibody scaffold (affibody). We demonstrated that the cell phenotype that depends on the avidity of displayed proteins can be controlled through titration against increasing tetracycline concentrations. The regulation of glucose oxidase enabled the quantitative encapsulation of single cells as a high-throughput screening method for directed evolution; the titration of an XDocIII domain led to the control of cell adhesion under shear stress; and the control of the affibody binding to PD-L1 allowed miniaturized affinity-constant measurements, showcasing the applicability of the yeast titratable display platform on protein engineering projects. Chapter 4 demonstrates: the development of a strategy to evolve new binding functions in a mechanostable protein; the engineering of a non-antibody scaffold based on bacterial cohesins with robust stability; and the creation and characterization of anti-PD-L1 Mechanobodies for immune checkpoint therapy. We implemented a semi-rational methodology to construct ligand-binding non-antibody scaffolds that withstand denaturing mechanical forces. By coupling yeast display to bioinformatic and biophysical mutational studies on the seventh cohesin domain Coh7, we selected optimized positions in two loops for library generation. We employed high-throughput screening methods compatible with yeast display to create the first alternative scaffold with a novel binding function to PD-L1 for enhanced immunotherapy, denoted mechanobody. After protein engineering and discovery rounds, we isolated a pool of mechanobodies with two-digit nanomolar dissociation constants, demonstrating the approach's utility. The best lead hit showed the PD-L1 binding motif 86LHGFY90 and 126NHDPR130. Chapter 5 offers the drawn conclusions for the project and an overview of the foreseen research avenues for each developed system. In addition, the appendices contain secondary publications from the author’s collaborations to study protein functions in medically relevant bacterial adhesins and develop hemostatic biopolymers. Altogether, the results outlined here pinpoint the relevance and opportunities of yeast display to engineer synthetic systems and molecules with novel functions in biotechnological research. We hope that this research will contribute to a deeper understanding of yeast display and enable further steps for biotechnological advances.
Advisors:Nash , Michael
Committee Members:Ricklin, Daniel and Correia, Bruno
Faculties and Departments:05 Faculty of Science > Departement Chemie > Chemie > Synthetic Systems (Nash)
05 Faculty of Science > Departement Pharmazeutische Wissenschaften > Pharmazie > Molecular Pharmacy (Ricklin)
UniBasel Contributors:Nash, Michael and Ricklin, Daniel
Item Type:Thesis
Thesis Subtype:Doctoral Thesis
Thesis no:15419
Thesis status:Complete
Number of Pages:202
Language:English
Identification Number:
  • urn: urn:nbn:ch:bel-bau-diss154195
edoc DOI:
Last Modified:31 Jul 2024 04:30
Deposited On:30 Jul 2024 11:13

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