Biology subjects

Zelada, A. M.

Publications and source records attributed to Zelada, A. M..

3 recordsLinked to original sources

bioRxiv2021

Production of a Highly Immunogenic Antigen from SARS-CoV-2 by Covalent Coupling of the Receptor Binding Domain of Spike Protein to a Multimeric Carrier

Since the discovery of SARS-CoV-2, several antigens have been proposed to be part of COVID-19 vaccines. The receptor binding domain (RBD) of Spike protein is one of the promising candidates to develop effective vaccines since it can induce potent neutralizing antibodies. We previously reported the production of RBD in Pichia pastoris and showed it is structurally identical to the protein produced in mammalian HEK-293T cells. In this work we designed an RBD multimer construct with the purpose of increasing RBD immunogenicity. We produced multimeric particles by a transpeptidation reaction between the RBD expressed in P. pastoris and Lumazine Synthase from Brucella abortus (BLS), which is a highly immunogenic and very stable decameric protein of 170 kDa. We vaccinated mice with two doses 30 days apart, and then we measured humoral immune response. When the number of RBD copies coupled to BLS was high (6-7 RBD molecules per BLS decamer, in average), the immune response was significantly better than that elicited by RBD alone or even by RBD-BLS comprising low number of RBD copies (1-2 RBD molecules per BLS decamer). Remarkably, the construct with high number of RBD copies induced high IgG titers with high neutralizing capacity. Furthermore, a superior immune response was observed when Al(OH)3 adjuvant was added to this formulation, exhibiting a higher titer of neutralizing antibodies. Altogether our results suggest that RBD covalent coupled to BLS forming a multimer-particle shows an advantageous architecture to the antigen-presentation to the immune system which enhances immune responses. This new antigen should be considered a potent candidate for a protein-based vaccine.

biochemistry↗

bioRxiv2021

Genomic diversification of dehydrin gene family in vascular plants: three distinctive orthologue groups and a novel KS-dehydrin conserved protein motif.

Dehydrins (DHNs) are a family of plant proteins that play important roles on abiotic stress tolerance and seed development. They are classified into five structural subgroups: K-, SK-, YK-, YSK-, and KS-DHNs, according to the presence of conserved motifs named K-, Y- and S-segments.We carried out a comparative structural and phylogenetic analysis of these proteins, focusing on the less-studied KS-type DHNs. A search for conserved motifs in DHNs from 56 plant genomes revealed that KS-DHNs possess a unique and highly conserved N-terminal, 15-residue amino acid motif not previously described. This novel motif, that we named H-segment, is present in DHNs of angiosperms, gymnosperms and lycophytes, suggesting that HKS-DHNs were present in the first vascular plants. Phylogenetic and microsynteny analyses indicate that the five structural subgroups of angiosperm DHNs can be assigned to three groups of orthologue genes, characterized by the presence of the H-, F- or Y-segments. Importantly, the hydrophilin character of DHNs correlate with the phylogenetic origin of the DHNs rather than to the traditional structural subgroups. We propose that angiosperm DHNs can be ultimately subdivided into three orthologous groups, a phylogenetic framework that should help future studies on the evolution and function of this protein family.

plant biology↗

bioRxiv2020

Structural and Functional Comparison of SARS-CoV-2-Spike Receptor Binding Domain Produced in Pichia pastoris and Mammalian Cells

The yeast Pichia pastoris is a cost-effective and easily scalable system for recombinant protein production. In this work we compared the conformation of the receptor binding domain (RBD) from SARS-CoV-2 Spike protein expressed in P. pastoris and in the well established HEK-293T mammalian cell system. RBD obtained from both yeast and mammalian cells was properly folded, as indicated by UV-absorption, circular dichroism and tryptophan fluorescence. They also had similar stability, as indicated by temperature-induced unfolding (observed Tm were 50 {degrees}C and 52 {degrees}C for RBD produced in P. pastoris and HEK-293T cells, respectively). Moreover, the stability of both variants was similarly reduced when the ionic strength was increased, in agreement with a computational analysis predicting that a set of ionic interactions may stabilize RBD structure. Further characterization by HPLC, size-exclusion chromatography and mass spectrometry revealed a higher heterogeneity of RBD expressed in P. pastoris relative to that produced in HEK-293T cells, which disappeared after enzymatic removal of glycans. The production of RBD in P. pastoris was scaled-up in a bioreactor, with yields above 45 mg/L of 90% pure protein, thus potentially allowing large scale immunizations to produce neutralizing antibodies, as well as the large scale production of serological tests for SARS-CoV-2.

biochemistry↗