Biology subjects

Gasulla, J.

Publications and source records attributed to Gasulla, J..

3 recordsLinked to original sources

bioRxiv2022

Optimization and validation of a Protein Phosphatase inhibition assay for accessible microcystin detection.

The presence of cyanobacterial toxins in freshwater constitutes an increasing public health concern, especially affecting developing countries where the high cost of available methods makes monitoring programs difficult. The phosphatase inhibition assay (PPIAs) is a sensitive method with low instrument requirements that allows the quantification of the most frequent cyanotoxins, microcystins (MC). In this work, we implemented a PPIAs, starting from Protein Phosphatase 1 (PP1) expression up to the validation with samples of algal blooms from Argentina. To do this, we optimized the expression and lyophilization of PP1, and the assay conditions. Also, we included robustness and possible interfering analysis. We evaluated the most widely used cyanobacterial lysis methods and determined that heating for 15 minutes at 95{degrees}C is simple and adequate for this assay. Then, we performed MC spikes recovery assays on water samples from three dams from Argentina, resulting in a recovery ranging from 77 to 115%. The limit of detection (LOD) was 0.4 g/L and the linear range is 0.4 g/L - 5 g/L. Finally, we evaluated 64 environmental samples where MC was measured by ELISA test containing from 0 g/L to 625 g/L. The PPIA showed excellent correlation (Pearson correlation coefficient = 0.967), no false negative and no false positives above the 1 g/L WHO guideline (0.11 false positive rate). In conclusion, we optimized and validated a PPIAs to be an effective and accessible alternative to available commercial tests.

biochemistry↗

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↗

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↗