UC Davis

The use of engineered protein systems has become highly prevalent across a wide range of biological fields. In synthetic biology, engineered proteins and protein systems offer precise methods for regulation of cellular behavior, enabling high sensitivity, tunability, and temporal resolution. In bioproduction, proteins are engineered to have enhanced functional properties or other characteristics that facilitate production, purification, and storage. The work in this dissertation explores some of this vast landscape through several chapters that discuss the implementation of optogenetics in Chinese Hamster Ovary (CHO) cells and computationally guided design and production of higher performing severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) truncations.

In synthetic biology, many genetic, chemical, and environmental approaches have been developed to modulate cellular pathways to improve titers. However, these methods are often irreversible or have off-target effects. Development of synthetic biology techniques which are precise, tunable, and reversible will facilitate temporal regulation of target pathways to maximize titers and protein quality. In this study, we investigate the use of optogenetics in CHO cells. Chinese hamster ovary (CHO) cells are widely used for industrial production of biopharmaceuticals. The light-activated CRISPR-dCas9 effector (LACE) system was first transiently transfected to express eGFP in a light-inducible manner. Then, a stable system was tested using lentiviral transduction. Transient transfections resulted in increasing eGFP expression as a function of LED intensity, and fluorescence decreased once the LACE system was deactivated.

Optogenetic control of cellular pathways and gene circuits in mammalian cells is a new frontier in mammalian genetic engineering, and protein engineering has significantly increased the performance of these systems. As a low-cost, tunable, and reversible input, light is highly adept at spatiotemporal, orthogonal regulation of cellular behavior, advancing applications such as protein bioproduction and cultivated meat. However, light is absorbed and scattered as it travels through media and cells, and the applicability of optogenetics in larger mammalian bioreactors has not been determined. In this work, we computationally explore the size limit to which optogenetics can be applied in cylindrical bioreactors at relevant height-to-diameter ratios for mammalian cell culture. We model the propagation of light using the radiative transfer equation and consider changes in reactor volume, absorption coefficient, scattering coefficient, and scattering anisotropy. We observed sufficient light penetration for activation for bioreactor sizes of up to 80,000 L with maximal cell densities, with decreasing efficiency for larger bioreactors. For a 100,000 L bioreactor, we determined that lower cell densities of up to 1.5⋅107 cells/mL can be supported. We conclude that optogenetics can be applied to bioreactors at an industrial scale and may be a valuable tool for specific biomanufacturing applications.

Rather than using engineered proteins to regulate cellular behavior, protein engineering can also be performed to directly modify proteins of interest for enhanced bioproduction. During the SARS-CoV-2 pandemic, protein engineering has played a major role in Spike protein bioproduction. Spike is a key protein that mediates viral entry into cells and elicits antibody responses. Its importance in infection, diagnostics, and vaccinations has created a large demand for purified Spike for diagnostic, clinical and research applications. Spike is difficult to express, prompting modifications to the protein and expression platforms to improve yields. Alternatively, Spike receptor binding domain (RBD) is commonly expressed with higher titers, though it has lower sensitivity in serological assays. Engineered Spike proteins for higher stability have greatly increased expression levels, which is critical for rapid, cost-effective production. We first improve transient Spike expression in Chinese hamster ovary (CHO) cells and demonstrate that Spike titers increase significantly over longer expression periods compared to RBD. Next, we developed 8 Spike truncations in pursuit of a truncation with both high expression and antibody binding. Truncations were designed such that the RBD sequence was conserved, and truncation points do not interrupt major secondary structures. Two truncations had higher expression than RBD, and one truncation had higher affinity to antibodies than did Spike. Binding of one truncation, T1, to ACE2-Fc was comparable to that of RBD, and N-linked glycosylation profiles resembled those of RBD and Spike very closely. These results suggest T1 is a promising Spike alternative for use in various applications.