tetano
Editor, Senior Moderator
Biochim Biophys Acta Gen Subj
. 2021 Mar 14;129893.
doi: 10.1016/j.bbagen.2021.129893. Online ahead of print.
Genetic modification to design a stable yeast-expressed recombinant SARS-CoV-2 receptor binding domain as a COVID-19 vaccine candidate
Wen-Hsiang Chen[SUP] 1 [/SUP], Junfei Wei[SUP] 2 [/SUP], Rakhi Tyagi Kundu[SUP] 2 [/SUP], Rakesh Adhikari[SUP] 2 [/SUP], Zhuyun Liu[SUP] 2 [/SUP], Jungsoon Lee[SUP] 2 [/SUP], Leroy Versteeg[SUP] 2 [/SUP], Cristina Poveda[SUP] 2 [/SUP], Brian Keegan[SUP] 2 [/SUP], Maria Jose Villar[SUP] 2 [/SUP], Ana C de Araujo Leao[SUP] 2 [/SUP], Joanne Altieri Rivera[SUP] 2 [/SUP], Portia M Gillespie[SUP] 2 [/SUP], Jeroen Pollet[SUP] 1 [/SUP], Ulrich Strych[SUP] 1 [/SUP], Bin Zhan[SUP] 1 [/SUP], Peter J Hotez[SUP] 3 [/SUP], Maria Elena Bottazzi[SUP] 4 [/SUP]
Affiliations
Abstract
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.
. 2021 Mar 14;129893.
doi: 10.1016/j.bbagen.2021.129893. Online ahead of print.
Genetic modification to design a stable yeast-expressed recombinant SARS-CoV-2 receptor binding domain as a COVID-19 vaccine candidate
Wen-Hsiang Chen[SUP] 1 [/SUP], Junfei Wei[SUP] 2 [/SUP], Rakhi Tyagi Kundu[SUP] 2 [/SUP], Rakesh Adhikari[SUP] 2 [/SUP], Zhuyun Liu[SUP] 2 [/SUP], Jungsoon Lee[SUP] 2 [/SUP], Leroy Versteeg[SUP] 2 [/SUP], Cristina Poveda[SUP] 2 [/SUP], Brian Keegan[SUP] 2 [/SUP], Maria Jose Villar[SUP] 2 [/SUP], Ana C de Araujo Leao[SUP] 2 [/SUP], Joanne Altieri Rivera[SUP] 2 [/SUP], Portia M Gillespie[SUP] 2 [/SUP], Jeroen Pollet[SUP] 1 [/SUP], Ulrich Strych[SUP] 1 [/SUP], Bin Zhan[SUP] 1 [/SUP], Peter J Hotez[SUP] 3 [/SUP], Maria Elena Bottazzi[SUP] 4 [/SUP]
Affiliations
- PMID: 33731300
- DOI: 10.1016/j.bbagen.2021.129893
Abstract
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.