Date of Graduation

5-2024

Document Type

Dissertation

Degree Name

Doctor of Philosophy in Engineering (PhD)

Degree Level

Graduate

Department

Chemical Engineering

Advisor/Mentor

Beitle, Robert R. Jr.

Committee Member

Hestekin, Christa N.

Second Committee Member

Pinto, Ines

Third Committee Member

Servoss, Shannon L.

Fourth Committee Member

Lyle, Chris

Keywords

Yeast surface display; SARS-CoV-2; Michaelis-Menten model

Abstract

The arming of yeast cell wall, also known as yeast surface display, is an effective technology for expressing target proteins extracellularly. It has been shown to provide bioengineered yeast with qualities that are not known to native yeast such as the ability to survive harsh conditions including high pH and temperature, for example. Furthermore, this technique has been implemented for various applications which include, protein engineering, bioethanol production, bioremediation, vaccine construction, and antibody development. The major mechanisms of yeast surface display exploit agglutination, flocculation, or proteins with internal repeats as the means by which a target (protein) is covalently attached to the cell wall surface. The most commonplace and successfully utilized amongst the types are agglutination due to its advantages over the others which include the ease of genetic manipulation to extend the amino or carboxyl terminus of the secretory sequence. This advantage circumvents the problem of impeding the functional and active domains of displayed target proteins. In this first part of this project, DNA for the receptor binding domain of SARS-CoV-2 virus was inserted into pYD1 expression vector, which led to display on the cell wall of Saccharomyces cerevisiae. Culturing and induction of this bioengineered yeast in galactose medium was shown to display the target sequence. Furthermore, the yeast clones were used as the test line in a lateral flow immunoassay to detect COVID antibody in patient sera. The first part of the work resulted in a successful, cost-effective technique for the construction of COVID test standards that did not rely on complicated downstream techniques to isolate the material The second part of this project expanded the work to include a kinetic description of heterologous protein display. A simple structured model based on limited respiratory capacity was extended to recombinant yeast to describe substrate uptake, ethanol production, biomass growth, protein production, and surface display of heterologous proteins. Surface display is described as a second order kinetic reaction which was dependent on the concentrations of cytoplasmic (periplasmic) proteins and yeast concentration. The description was validated with data from literature and experiments. Thus, the second part of this work was also shown to follow a simplified Michaelis-Menten model since the second order form was useful to describe surface display. Lastly, this dissertation developed a procedure to immobilize any recombinant protein to the cell surface via Aga1 – Aga 2 interactions. E. coli expressed proteins were attached to the cell will using a mild oxidation buffer which reduces Cys, forming disulphides between Aga1 and Aga2-extended proteins. With the knowledge that EBY100 yeast cell wall is composed of 104-105 agglutin adhesion protein, should these be unoccupied by other (native) proteins, a novel strategy for immobilization emerges.

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