High-yield Production in Escherichia Coli and Convenient Purification of a Candidate Vaccine Against SARS-CoV-2

Overview

Journal Biotechnol Lett

Specialties Biochemistry
Biotechnology

Date 2022 Sep 26

PMID 36161539

Authors

Giulia Maltoni

Lorenzo Scutteri

Francesca Mensitieri

Fabrizio Dal Piaz

Alejandro Hochkoeppler

Affiliations

Soon will be listed here.

Abstract

Objectives: The aim of the present work was to identify a time-saving, effective, and low-cost strategy to produce in Escherichia coli a protein chimera representing a fusion anti-SARS-CoV-2 candidate vaccine, consisting of immunogenic and antigenic moieties.

Results: We overexpressed in E. coli BL21(DE3) a synthetic gene coding for CRM197-RBD, and the target protein was detected in inclusion bodies. CRM197-RBD was solubilized with 1 % (w/v) of the anionic detergent N-lauroylsarcosine (sarkosyl), the removal of which from the protein solution was conveniently accomplished with Amberlite XAD-4. The detergent-free CRM197-RBD was then separated from contaminating DNA using polyethylenimine (PEI), and finally purified from PEI by salting out with ammonium sulfate. Structural (CD spectrum) and functional (DNase activity) assays revealed that the CRM197-RBD chimera featured a native and active conformation. Remarkably, we determined a yield of purified CRM197-RBD equal to 23 mg per litre of culture.

Conclusions: To produce CRM197-RBD, we devised the use of sarkosyl as an alternative to urea to solubilize the target protein from E. coli inclusion bodies, and the easy removal of sarkosyl by means of Amberlite XAD-4.

Citing Articles

Sodium Dodecyl Sulfate Analogs as a Potential Molecular Biology Reagent.

Arakawa T, Niikura T, Kita Y, Akuta T Curr Issues Mol Biol. 2024; 46(1):621-633.

PMID: 38248342 PMC: 10814491. DOI: 10.3390/cimb46010040.

Challenges and Opportunities in the Process Development of Chimeric Vaccines.

Chauhan S, Khasa Y Vaccines (Basel). 2023; 11(12).

PMID: 38140232 PMC: 10747103. DOI: 10.3390/vaccines11121828.

References

Bellone M, Puglisi A, Dal Piaz F, Hochkoeppler A . Production in Escherichia coli of recombinant COVID-19 spike protein fragments fused to CRM197. Biochem Biophys Res Commun. 2021; 558:79-85. PMC: 8057744. DOI: 10.1016/j.bbrc.2021.04.056. View

Bradford M . A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976; 72:248-54. DOI: 10.1016/0003-2697(76)90527-3. View

Bruce C, Baldwin R, Lessnick S, Wisnieski B . Diphtheria toxin and its ADP-ribosyltransferase-defective homologue CRM197 possess deoxyribonuclease activity. Proc Natl Acad Sci U S A. 1990; 87(8):2995-8. PMC: 53820. DOI: 10.1073/pnas.87.8.2995. View

Conte E, Vincelli G, Schaaper R, Bressanin D, Stefan A, Dal Piaz F . Stabilization of the Escherichia coli DNA polymerase III ε subunit by the θ subunit favors in vivo assembly of the Pol III catalytic core. Arch Biochem Biophys. 2012; 523(2):135-43. PMC: 3441146. DOI: 10.1016/j.abb.2012.04.013. View

Cui Z, Liu P, Wang N, Wang L, Fan K, Zhu Q . Structural and functional characterizations of infectivity and immune evasion of SARS-CoV-2 Omicron. Cell. 2022; 185(5):860-871.e13. PMC: 8786603. DOI: 10.1016/j.cell.2022.01.019. View

Dimitrov J, Lacroix-Desmazes S, Kaveri S . Important parameters for evaluation of antibody avidity by immunosorbent assay. Anal Biochem. 2011; 418(1):149-51. DOI: 10.1016/j.ab.2011.07.007. View

Elena C, Ravasi P, Castelli M, Peiru S, Menzella H . Expression of codon optimized genes in microbial systems: current industrial applications and perspectives. Front Microbiol. 2014; 5:21. PMC: 3912506. DOI: 10.3389/fmicb.2014.00021. View

McGuire B, Mela J, Thompson V, Cucksey L, Stevens C, McWhinnie R . Escherichia coli recombinant expression of SARS-CoV-2 protein fragments. Microb Cell Fact. 2022; 21(1):21. PMC: 8817660. DOI: 10.1186/s12934-022-01753-0. View

Porro M, Saletti M, Nencioni L, Tagliaferri L, Marsili I . Immunogenic correlation between cross-reacting material (CRM197) produced by a mutant of Corynebacterium diphtheriae and diphtheria toxoid. J Infect Dis. 1980; 142(5):716-24. DOI: 10.1093/infdis/142.5.716. View

10.

Rappuoli R, De Gregorio E, Costantino P . On the mechanisms of conjugate vaccines. Proc Natl Acad Sci U S A. 2018; 116(1):14-16. PMC: 6320500. DOI: 10.1073/pnas.1819612116. View

11.

Shang J, Ye G, Shi K, Wan Y, Luo C, Aihara H . Structural basis of receptor recognition by SARS-CoV-2. Nature. 2020; 581(7807):221-224. PMC: 7328981. DOI: 10.1038/s41586-020-2179-y. View

12.

Tauzin A, Gendron-Lepage G, Nayrac M, Anand S, Bourassa C, Medjahed H . Evolution of Anti-RBD IgG Avidity following SARS-CoV-2 Infection. Viruses. 2022; 14(3). PMC: 8949389. DOI: 10.3390/v14030532. View

13.

Uchida T, PAPPENHEIMER Jr A, Greany R . Diphtheria toxin and related proteins. I. Isolation and properties of mutant proteins serologically related to diphtheria toxin. J Biol Chem. 1973; 248(11):3838-44. View

14.

Zhang J, Cai Y, Lavine C, Peng H, Zhu H, Anand K . Structural and functional impact by SARS-CoV-2 Omicron spike mutations. Cell Rep. 2022; 39(4):110729. PMC: 8995406. DOI: 10.1016/j.celrep.2022.110729. View