Genetic modification to design a stable yeast-expressed recombinant SARS-CoV-2 receptor binding domain as a COVID-19 vaccine candidate

Biochim Biophys Acta Gen Subj. 2021 Jun;1865(6):129893. doi: 10.1016/j.bbagen.2021.129893. Epub 2021 Mar 14.


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.

Keywords: Biophysical characterization; Biotechnology; Coronavirus; P. pastoris.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • COVID-19 Vaccines / immunology*
  • Cloning, Molecular
  • Gene Expression
  • Protein Domains
  • Protein Structure, Tertiary
  • Recombinant Proteins / chemistry*
  • Recombinant Proteins / genetics*
  • SARS-CoV-2 / genetics*
  • SARS-CoV-2 / immunology*
  • Saccharomycetales / genetics*
  • Spike Glycoprotein, Coronavirus / chemistry
  • Spike Glycoprotein, Coronavirus / genetics*
  • Spike Glycoprotein, Coronavirus / immunology


  • COVID-19 Vaccines
  • Recombinant Proteins
  • Spike Glycoprotein, Coronavirus
  • spike protein, SARS-CoV-2

Supplementary concepts

  • Komagataella pastoris