期刊
BIOCHIMICA ET BIOPHYSICA ACTA-GENERAL SUBJECTS
卷 1865, 期 6, 页码 -出版社
ELSEVIER
DOI: 10.1016/j.bbagen.2021.129893
关键词
Coronavirus; P. pastoris; Biophysical characterization; Biotechnology
资金
- Robert J. Kleberg Jr. and Helen C. Kleberg Foundation, USA
- Fifth Generation, Inc. (Tito's Handmade Vodka), USA
- JPB Foundation, USA
- National Institute of Health-National Institute of Allergy and Infectious Diseases (NIHNIAHID), USA [AI14087201]
- Texas Children's Hospital Center for Vaccine Development Intramural Funds, USA
The study successfully demonstrated that genetic modification can lead to a better-controlled and more stable vaccine candidate, which is crucial for the production of biologics or drugs for human use.
Background: Coronavirus disease 2019 (COVID-19) caused by SARS-CoV-2 has now spread worldwide to infect over 110 million people, with approximately 2.5 million reported deaths. A safe and effective vaccine remains urgently needed. Method: We constructed three variants of the recombinant receptor-binding domain (RBD) of the SARS-CoV-2 spike (S) protein (residues 331-549) in yeast as follows: (1) a wild type RBD (RBD219-WT), (2) a deglycosylated form (RBD219-N1) by deleting the first N-glycosylation site, and (3) a combined deglycosylated and cysteine-mutagenized form (C538A-mutated variant (RBD219-N1C1)). We compared the expression yields, biophysical characteristics, and functionality of the proteins produced from these constructs. Results and conclusions: These three recombinant RBDs showed similar secondary and tertiary structure thermal stability and had the same affinity to their receptor, angiotensin-converting enzyme 2 (ACE-2), suggesting that the selected deletion or mutations did not cause any significant structural changes or alteration of function. However, RBD219-N1C1 had a higher fermentation yield, was easier to purify, was not hyperglycosylated, and had a lower tendency to form oligomers, and thus was selected for further vaccine development and evaluation. General significance: By genetic modification, we were able to design a better-controlled and more stable vaccine candidate, which is an essential and important criterion for any process and manufacturing of biologics or drugs for human use.
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