Lactococcus lactis bacterium can be genetically modified to transport the spike protein from SARS-CoV-2, making it a potential candidate for a COVID-19 mucosal vaccine. This study aimed to optimize the nisin concentration, pH, incubation time, and media composition to induce spike protein expression. The concentrations of nisin used in this study ranged from 0 to 40 ng/mL, the incubation period was 3 to 24 hr, and the pH of the growth media ranged from 4 to 8. The media was also supplemented with various yeast extract and sucrose concentrations. The highest protein band intensity was observed at a concentration of 40 ng/mL and an incubation period of 9 hr. Supplementation with 4% w/v yeast extract and 6% w/v sucrose significantly increased the expression of HCR spike protein. In silico simulation suggested a maximal protein band intensity of 70.95 arbitrary units, while the nisin concentration needed to produce half the maximal protein band intensity was estimated to be 9.599 ng/mL. No significant difference in spike protein expression was found between pH variations. The media composition, inducer, and incubation time strongly affect the spike protein expression.

Department of Artificial Intelligence Institute of Computer Science of the Czech Academy of Sciences Pod Vodarenskou vezi 271 2 182 00 Praha 8 Liben Czech Republic

Department of Biophysics and Physical Chemistry Faculty of Pharmacy in Hradec Kralove Charles University Akademika Heyrovského 1203 8 500 03 Hradec Králové Czech Republic

Department of Pharmacy Medical Faculty Universitas Brawijaya Jalan Veteran Malang 65145 East Java Indonesia

Laboratory of Molecular Biotechnology Institute for Aqua Regeneration Shinshu University 8304 Minamiminowa Kamiina Nagano 399 4598 Japan

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