tetano
Editor, Senior Moderator
Trop Med Infect Dis
. 2023 Nov 16;8(11):501.
doi: 10.3390/tropicalmed8110501. Development of an Optimized Process for Functional Recombinant SARS-CoV-2 Spike S1 Receptor-Binding Domain Protein Produced in the Baculovirus Expression Vector System
Mohamed Boumaiza[SUP] 1 [/SUP], Ameni Chaabene[SUP] 1 [/SUP], Ines Akrouti[SUP] 1 [/SUP], Meriem Ben Zakour[SUP] 1 [/SUP], Hana Askri[SUP] 1 [/SUP], Said Salhi[SUP] 1 [/SUP], Wafa Ben Hamouda[SUP] 2 [/SUP], Soumaya Marzouki[SUP] 2 [/SUP], Chaouki Benabdessalem[SUP] 2 [/SUP], Melika Ben Ahmed[SUP] 2 [/SUP], Khaled Trabelsi[SUP] 1 [/SUP], Samia Rourou[SUP] 1 [/SUP]
Affiliations
To map the spread of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) and evaluate immune response variations against this virus, it is essential to set up efficient serological tests locally. The SARS-CoV-2 immunogenic proteins were very expensive and not affordable for lower- middle-income countries (LMICs). For this purpose, the commonly used antigen, receptor-binding domain (RBD) of spike S1 protein (S1RBD), was produced using the baculovirus expression vector system (BEVS). In the current study, the expression of S1RBD was monitored using Western blot under different culture conditions. Different parameters were studied: the multiplicity of infection (MOI), cell density at infection, and harvest time. Hence, optimal conditions for efficient S1RBD production were identified: MOI 3; cell density at infection 2-3 × 10[SUP]6[/SUP] cells/mL; and time post-infection (tPI or harvest time) of 72 h and 72-96 h, successively, for expression in shake flasks and a 7L bioreactor. A high production yield of S1RBD varying between 4 mg and 70 mg per liter of crude cell culture supernatant was achieved, respectively, in the shake flasks and 7L bioreactor. Moreover, the produced S1RBD showed an excellent antigenicity potential against COVID-19 (Wuhan strain) patient sera evaluated by Western blot. Thus, additional serological assays, such as in-house ELISA and seroprevalence studies based on the purified S1RDB, were developed.
Keywords: SARS-CoV-2; Sf9 cells; cell density at infection; multiplicity of infection; receptor-binding domain; spike S1 glycoprotein.
. 2023 Nov 16;8(11):501.
doi: 10.3390/tropicalmed8110501. Development of an Optimized Process for Functional Recombinant SARS-CoV-2 Spike S1 Receptor-Binding Domain Protein Produced in the Baculovirus Expression Vector System
Mohamed Boumaiza[SUP] 1 [/SUP], Ameni Chaabene[SUP] 1 [/SUP], Ines Akrouti[SUP] 1 [/SUP], Meriem Ben Zakour[SUP] 1 [/SUP], Hana Askri[SUP] 1 [/SUP], Said Salhi[SUP] 1 [/SUP], Wafa Ben Hamouda[SUP] 2 [/SUP], Soumaya Marzouki[SUP] 2 [/SUP], Chaouki Benabdessalem[SUP] 2 [/SUP], Melika Ben Ahmed[SUP] 2 [/SUP], Khaled Trabelsi[SUP] 1 [/SUP], Samia Rourou[SUP] 1 [/SUP]
Affiliations
- PMID: 37999620
- DOI: 10.3390/tropicalmed8110501
To map the spread of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) and evaluate immune response variations against this virus, it is essential to set up efficient serological tests locally. The SARS-CoV-2 immunogenic proteins were very expensive and not affordable for lower- middle-income countries (LMICs). For this purpose, the commonly used antigen, receptor-binding domain (RBD) of spike S1 protein (S1RBD), was produced using the baculovirus expression vector system (BEVS). In the current study, the expression of S1RBD was monitored using Western blot under different culture conditions. Different parameters were studied: the multiplicity of infection (MOI), cell density at infection, and harvest time. Hence, optimal conditions for efficient S1RBD production were identified: MOI 3; cell density at infection 2-3 × 10[SUP]6[/SUP] cells/mL; and time post-infection (tPI or harvest time) of 72 h and 72-96 h, successively, for expression in shake flasks and a 7L bioreactor. A high production yield of S1RBD varying between 4 mg and 70 mg per liter of crude cell culture supernatant was achieved, respectively, in the shake flasks and 7L bioreactor. Moreover, the produced S1RBD showed an excellent antigenicity potential against COVID-19 (Wuhan strain) patient sera evaluated by Western blot. Thus, additional serological assays, such as in-house ELISA and seroprevalence studies based on the purified S1RDB, were developed.
Keywords: SARS-CoV-2; Sf9 cells; cell density at infection; multiplicity of infection; receptor-binding domain; spike S1 glycoprotein.