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1.
Protein Expr Purif ; 167: 105532, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-31711796

RESUMO

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.


Assuntos
Catepsina D/isolamento & purificação , Schistosoma mansoni/enzimologia , Animais , Ácido Aspártico Proteases/biossíntese , Ácido Aspártico Proteases/química , Ácido Aspártico Proteases/isolamento & purificação , Ácido Aspártico Proteases/metabolismo , Catepsina D/biossíntese , Catepsina D/química , Catepsina D/metabolismo , Catepsinas/biossíntese , Catepsinas/química , Catepsinas/isolamento & purificação , Catepsinas/metabolismo , Cromatografia em Gel , Dimerização , Células HEK293 , Humanos , Cinética , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Ultracentrifugação/métodos
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