RESUMEN
SARS-CoV-2 Nucleocapsid (N) is the most abundant viral protein expressed in host samples and is an important antigen for diagnosis. N is a 45 kDa protein that does not present disulfide bonds. Intending to avoid non-specific binding of SARS-CoV-2 N to antibodies from patients who previously had different coronaviruses, a 35 kDa fragment of N was expressed without a conserved motif in E. coli as inclusion bodies (N122-419-IB). Culture media and IB washing conditions were chosen to obtain N122-419-IB with high yield (370 mg/L bacterial culture) and protein purity (90%). High pressure solubilizes protein aggregates by weakening hydrophobic and ionic interactions and alkaline pH promotes solubilization by electrostatic repulsion. The association of pH 9.0 and 2.4 kbar promoted efficient solubilization of N122-419-IB without loss of native-like tertiary structure that N presents in IB. N122-419 was refolded with a yield of 85% (326 mg/L culture) and 95% purity. The refolding process takes only 2 hours and the protein is ready for use after pH adjustment, avoiding the necessity of dialysis or purification. Antibody binding of COVID-19-positive patients sera to N122-419 was confirmed by Western blotting. ELISA using N122-419 is effective in distinguishing between sera presenting antibodies against SARS-CoV-2 from those who do not. To the best of our knowledge, the proposed condition for IB solubilization is one of the mildest described. It is possible that the refolding process can be extended to a wide range of proteins with high yields and purity, even those that are sensible to very alkaline pH.
Asunto(s)
Anticuerpos Antivirales/sangre , Antígenos Virales/química , COVID-19/sangre , COVID-19/diagnóstico , Proteínas de la Nucleocápside de Coronavirus/química , Inmunoglobulina G/sangre , Cuerpos de Inclusión/química , Replegamiento Proteico , SARS-CoV-2/inmunología , Anticuerpos Antivirales/inmunología , Antígenos Virales/inmunología , COVID-19/virología , Proteínas de la Nucleocápside de Coronavirus/inmunología , Ensayo de Inmunoadsorción Enzimática/métodos , Escherichia coli/genética , Escherichia coli/metabolismo , Humanos , Concentración de Iones de Hidrógeno , Presión Hidrostática , Inmunoglobulina G/inmunología , Fosfoproteínas/química , Fosfoproteínas/inmunología , Estructura Terciaria de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/inmunología , SolubilidadRESUMEN
During 2020, the COVID-19 pandemic affected almost 108 individuals. Quite a number of vaccines against COVID-19 were therefore developed, and a few recently received authorization for emergency use. Overall, these vaccines target specific viral proteins by antibodies whose synthesis is directly elicited or indirectly triggered by nucleic acids coding for the desired targets. Among these targets, the receptor binding domain (RBD) of COVID-19 spike protein (SP) does frequently occur in the repertoire of candidate vaccines. However, the immunogenicity of RBD per se is limited by its low molecular mass, and by a structural rearrangement of full-length SP accompanied by the detachment of RBD. Here we show that the RBD of COVID-19 SP can be conveniently produced in Escherichia coli when fused to a fragment of CRM197, a variant of diphtheria toxin currently used for a number of conjugated vaccines. In particular, we show that the CRM197-RBD chimera solubilized from inclusion bodies can be refolded and purified to a state featuring the 5 native disulphide bonds of the parental proteins, the competence in binding angiotensin-converting enzyme 2, and a satisfactory stability at room temperature. Accordingly, our observations provide compulsory information for the development of a candidate vaccine directed against COVID-19.
Asunto(s)
Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Escherichia coli , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/genética , SARS-CoV-2 , Glicoproteína de la Espiga del Coronavirus/química , Glicoproteína de la Espiga del Coronavirus/genética , Proteínas Bacterianas/biosíntesis , Proteínas Bacterianas/aislamiento & purificación , Secuencia de Bases , Vacunas contra la COVID-19/química , Vacunas contra la COVID-19/genética , Vacunas contra la COVID-19/inmunología , Escherichia coli/genética , Escherichia coli/metabolismo , Cuerpos de Inclusión/química , Cuerpos de Inclusión/metabolismo , Espectrometría de Masas , Modelos Moleculares , Replegamiento Proteico , Estabilidad Proteica , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/aislamiento & purificación , SARS-CoV-2/química , SARS-CoV-2/genética , SARS-CoV-2/inmunología , Glicoproteína de la Espiga del Coronavirus/biosíntesis , Glicoproteína de la Espiga del Coronavirus/aislamiento & purificación , Temperatura , Factores de TiempoRESUMEN
Sensitive and specific serology tests are essential for epidemiological and public health studies of COVID-19 and for vaccine efficacy testing. The presence of antibodies to SARS-CoV-2 surface glycoprotein (Spike) and, specifically, its receptor-binding domain (RBD) correlates with inhibition of SARS-CoV-2 binding to the cellular receptor and viral entry into the cells. Serology tests that detect antibodies targeting RBD have high potential to predict COVID-19 immunity and to accurately determine the extent of the vaccine-induced immune response. Cost-effective methods of expression and purification of Spike and its fragments that preserve antigenic properties are essential for development of such tests. Here we describe a method of production of His6-tagged S319-640 fragment containing RBD in E. coli. It includes expression of the fragment, solubilization of inclusion bodies, and on-the-column refolding. The antigenic properties of the resulting product are similar, but not identical to the RBD-containing fragment expressed in human cells.