Seongwon Jung’s research while affiliated with University of California, Davis and other places

SARS‐CoV‐2 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 clinical and research applications. Spike is difficult to express, prompting modifications to the protein and expression platforms to improve yields. Alternatively, the Spike receptor‐binding domain (RBD) is commonly expressed with higher titers, though it has lower sensitivity in serological assays. Here, we improve transient Spike expression in Chinese hamster ovary (CHO) cells. We demonstrate that Spike titers increase significantly over the expression period, maximizing at 14 mg/L on day 7. In comparison, RBD titers peak at 54 mg/L on day 3. Next, we develop 8 Spike truncations (T1‐T8) in pursuit of truncation with high expression and antibody binding. The truncations T1 and T4 express at 130 mg/L and 73 mg/L, respectively, which are higher than our RBD titers. Purified proteins were evaluated for binding to antibodies raised against full‐length Spike. T1 has similar sensitivity as Spike against a monoclonal antibody and even outperforms Spike for a polyclonal antibody. These results suggest that T1 is a promising Spike alternative for use in various applications. This article is protected by copyright. All rights reserved

SARS-CoV-2 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 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. Here, we improve transient Spike expression in Chinese hamster ovary (CHO) cells. We demonstrate that Spike titers increase significantly over the expression period, maximizing at 14 mg/L at day 7. In comparison, RBD titers peak at 54 mg/L at day 3. Next, we develop 8 Spike truncations (T1-T8) in pursuit of a truncation with high expression and antibody binding. The truncations T1 and T4 express at 130 mg/L and 73 mg/L, respectively, which are higher than our RBD titers. Purified proteins were evaluated for binding to antibodies raised against full-length Spike. T1 has similar sensitivity as Spike against a monoclonal antibody and even outperforms Spike for a polyclonal antibody. These results suggest T1 is a promising Spike alternative for use in various applications.

Highly detailed steered Molecular Dynamics simulations are performed on differently glycosylated receptor binding domains of the SARS-CoV-2 spike protein. The binding strength and the binding range increases with glycosylation. The interaction energy rises very quickly with pulling the proteins apart and only slowly drops at larger distances. We see a catch slip type behavior where interactions during pulling break and are taken over by new interactions forming. The dominant interaction mode are hydrogen bonds but Lennard-Jones and electrostatic interactions are relevant as well.

Highly detailed steered Molecular Dynamics simulations are performed on differently glycosylated receptor binding domains of the SARS-CoV-2 spike protein. The binding strength and the binding range increases with glycosylation. The interaction energy rises very quickly with pulling the proteins apart and only slowly drops at larger distances. We see a catch slip type behavior where interactions during pulling break and are taken over by new interactions forming. The dominant interaction mode are hydrogen bonds but Lennard-Jones and electrostatic interactions are relevant as well. Statement of Significance Glycosylation of the receptor binding domain of the Spike protein of SARS-CoV-2 as well as the ACE2 receptor leads to stronger and longer ranged binding interactions between the proteins. Particularly, at shorter distances the interactions are between residues of the proteins themselves whereas at larger distances these interactions are mediated by the glycans. Abstract Figure

Producing recombinant proteins in transgenic plant cell suspension cultures in bioreactors provides controllability, reproducibility, scalability, and low‐cost production, although low yields remain the major challenge. The studies on scaling‐up to pilot‐scale bioreactors, especially in conventional stainless‐steel stirred tank bioreactors (STB), to produce recombinant proteins in plant cell suspension cultures are very limited. In this study, we scaled‐up the production of rice recombinant butyrylcholinesterase (rrBChE), a complex hydrolase enzyme that can be used to prophylactically and therapeutically treat against organophosphorus nerve agents and pesticide exposure, from metabolically regulated transgenic rice cell suspension cultures in a 40‐L pilot‐scale STB. Employing cyclical operation together with a simplified‐process operation (controlling gas sparging rate rather than dissolved oxygen and allowing natural sugar depletion) identified in lab‐scale (5 L) bioreactor studies, we found a consistent maximum total active rrBChE production level of 46–58 µg/g fresh weight in four cycles over 82 days of semicontinuous operation. Additionally, maintaining the overall volumetric oxygen mass transfer coefficient (kLa) in the pilot‐scale STB to be equivalent to the lab‐scale STB improves the maximum total active rrBChE production level and the maximum volumetric productivity to 85 µg/g fresh weight and 387 µg L⁻¹ day⁻¹, respectively, which are comparable to the lab‐scale culture. Here, we demonstrate pilot‐scale bioreactor performance using a metabolically regulated transgenic rice cell culture for long‐term, reproducible, and sustained production of rrBChE.