| Author | Araujo-Montoya, B. O. | |
| Author | Senger, M. R. | |
| Author | Gomes, B. F. | |
| Author | Harris, G. | |
| Author | Owens, R. J. | |
| Author | Silva Jr., F. P. | |
| Access date | 2020-09-03T19:51:00Z | |
| Available date | 2020-09-03T19:51:00Z | |
| Document date | 2020 | |
| Citation | ARAUJO-MONTOYA, B. O. et al. Schistosoma mansoni cathepsin D1: Biochemical and biophysical characterization of the recombinant enzyme expressed in HEK293T cells. Protein Expression and Purification, v. 167, 105532, p. 1-5, 2020. | pt_BR |
| ISSN | 1046-5928 | pt_BR |
| URI | https://www.arca.fiocruz.br/handle/icict/43173 | |
| Language | eng | pt_BR |
| Publisher | Elsevier | pt_BR |
| Rights | restricted access | |
| Subject in Portuguese | Schistosoma | pt_BR |
| Subject in Portuguese | Dímeros | pt_BR |
| Subject in Portuguese | Catepsina D | pt_BR |
| Subject in Portuguese | Ultracentrifugação analítica | pt_BR |
| Title | Schistosoma mansoni cathepsin D1: Biochemical and biophysical characterization of the recombinant enzyme expressed in HEK293T cells | pt_BR |
| Type | Article | |
| DOI | 10.1016/j.pep.2019.105532 | |
| Abstract | Schistosomes express a variety of aspartyl proteases (APs) with distinct roles in the helminth pathophysiology,
among which degradation of host haemoglobin is key, since it is the main amino acid source for these parasites.
A cathepsin D-like AP from Schistosoma mansoni (SmCD1) has been used as a model enzyme for vaccine and drug
development studies in schistosomes and yet a reliable expression system for readily producing the recombinant
enzyme in high yield has not been reported. To contribute to further advancing the knowledge about this valuable antischistosomal target, we developed a transient expression system in HEK 293T mammalian cells and
performed a biochemical and biophysical characterization of the recombinant enzyme (rSmCD1). It was possible
to express a recombinant C-terminal truncated form of SmCD1 (rSmCD1ΔCT) and purify it with high yield
(16 mg/L) from the culture supernatant. When analysed by Size-Exclusion Chromatography and multi-angle
laser light scattering, rSmCD1ΔCT behaved as a dimer at neutral pH, which is unusual for cathepsins D, turning
into a monomer after acidification of the medium. Through analytical ultrancentrifugation, the dimer was
confirmed for free rSmCD1ΔCT in solution as well as stabilization of the monomer during interaction with
pepstatin. The mammalian cell expression system used here was able to produce rSmCD1ΔCT with high yields
allowing for the first time the characterization of important kinetic parameters as well as initial description of its
biophysical properties. | pt_BR |
| Affilliation | Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Bioquímica Experimental e Computacional de Fármacos. Rio de Janeiro, RJ, Brasil. | pt_BR |
| Affilliation | Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Bioquímica Experimental e Computacional de Fármacos. Rio de Janeiro, RJ, Brasil. | pt_BR |
| Affilliation | Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Bioquímica Experimental e Computacional de Fármacos. Rio de Janeiro, RJ, Brasil. | pt_BR |
| Affilliation | Research Complex at Harwell, R92 Rutherford Appleton Laboratory. Didcot, UK | pt_BR |
| Affilliation | Research Complex at Harwell, R92 Rutherford Appleton Laboratory. Didcot, UK / Henry Wellcome Building for Genomic Medicine. The Division of Structural Biology. Oxford, UK. | pt_BR |
| Affilliation | Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Bioquímica Experimental e Computacional de Fármacos. Rio de Janeiro, RJ, Brasil. | pt_BR |
| Subject | Cathepsin D | pt_BR |
| Subject | Schistosome | pt_BR |
| Subject | HEK 293T cells | pt_BR |
| Subject | Analytical ultracentrifugation | pt_BR |
| Subject | SEC-MALS | pt_BR |
| Subject | Dimer | pt_BR |
| Embargo date | 2022-01-01 | |
