Production of a functionally active recombinant SARS-CoV-2 (COVID-19) 3C-Like protease and a soluble inactive 3C-like protease-RBD chimeric in a prokaryotic expression system
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Abstract
During the SARS-CoV-2 intracellular life-cycle, two large polyproteins, pp1a and pp1ab, are produced. Processing of these by viral cysteine proteases, the papain-like protease (PLpro) and the chymotrypsin-like 3C-like protease (3CL-pro) release non-structural proteins necessary for the establishment of the viral replication and transcription complex (RTC), crucial for viral replication. Hence, these proteases are considered prime targets against which anti-COVID-19 drugs could be developed. Here, we describe the expression of a highly soluble and functionally active recombinant 3CL-pro using Escherichia coli BL21 cells. In addition, we assessed the ability of our 3CL-pro to function as a carrier for the Receptor Binding Domain (RBD) of the Spike protein. The co-expressed chimeric protein, 3CLpro-RBD, did not exhibit 3CL-pro activity, but its enhanced solubility made purification easier and improved RBD antigenicity when tested against serum from vaccinated individuals in ELISAs. When used to immunise mice, the 3CLpro-RBD chimer elicited antibodies mainly to the 3CL-pro portion of the molecule indicating that a different chimeric composition (i.e., RBD/full Spike-3CLpro) or expression system (i.e., mammalian cells), might be required to produce and deliver a RBD with immunogenicity similar to the native protein. Chimeric proteins containing the 3CL-pro could represent an innovative approach to developing new COVID-19 vaccines.
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SciScore for 10.1101/2022.03.25.485815: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Ethics IRB: Ethical statement: Human experimental work was conducted according to Human Research Ethics Committees.
Consent: All participants provided written informed consent prior to the study.Sex as a biological variable Assessment of the immunogenicity of the r3CL-pro, the rRBD and the chimeric r3CLpro-RBD: Seven weeks-old male and female CD1 outbred mice were used to assess the immunogenicity of the recombinantly-produced proteins according to the schedule shown in Figure 2. Randomization not detected. Blinding not detected. Power Analysis not detected. Table 2: Resources
Antibodies Sentences Resources After 1 h incubation at RT, and washing five times with PBST, the secondary antibody HRP anti-Human IgG … SciScore for 10.1101/2022.03.25.485815: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Ethics IRB: Ethical statement: Human experimental work was conducted according to Human Research Ethics Committees.
Consent: All participants provided written informed consent prior to the study.Sex as a biological variable Assessment of the immunogenicity of the r3CL-pro, the rRBD and the chimeric r3CLpro-RBD: Seven weeks-old male and female CD1 outbred mice were used to assess the immunogenicity of the recombinantly-produced proteins according to the schedule shown in Figure 2. Randomization not detected. Blinding not detected. Power Analysis not detected. Table 2: Resources
Antibodies Sentences Resources After 1 h incubation at RT, and washing five times with PBST, the secondary antibody HRP anti-Human IgG (Fc specific) (Sigma-Aldrich) was added (1:15,000), and the plates incubated for 1 h at RT. anti-Human IgGsuggested: NoneAfter washing five times with PBST, the secondary antibody HRP goat to mouse-anti-IgG (ThermoFisher Scientific) was added (1:10,000), and the plates incubated for 1 h at RT. mouse-anti-IgGsuggested: NoneExperimental Models: Organisms/Strains Sentences Resources Assessment of the immunogenicity of the r3CL-pro, the rRBD and the chimeric r3CLpro-RBD: Seven weeks-old male and female CD1 outbred mice were used to assess the immunogenicity of the recombinantly-produced proteins according to the schedule shown in Figure 2. CD1suggested: NoneRecombinant DNA Sentences Resources Recombinant protein production in Escherichia coli cells and purification: Sequences encoding the 3CL-pro and RBD proteins were codon optimized for expression in Escherichia coli and cloned into the pET-28a(+) vector (Genscript Biotech). pET-28a(+ )suggested: NoneResults from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).
Results from LimitationRecognizer: An explicit section about the limitations of the techniques employed in this study was not found. We encourage authors to address study limitations.Results from TrialIdentifier: No clinical trial numbers were referenced.
Results from Barzooka: We did not find any issues relating to the usage of bar graphs.
Results from JetFighter: We did not find any issues relating to colormaps.
Results from rtransparent:- Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
- Thank you for including a funding statement. Authors are encouraged to include this statement when submitting to a journal.
- No protocol registration statement was detected.
Results from scite Reference Check: We found no unreliable references.
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