Expression, purification and functional verification of recombinant human α-galactosidase A in suspension CHO-S / 中国药理学通报
Chinese Pharmacological Bulletin
;
(12): 774-781, 2023.
Artigo
em Chinês
| WPRIM
| ID: wpr-1013819
ABSTRACT
Aim To express and purify rhα-Gal A with a 6 X His tag via using a serum-free expression system in high-density suspension culture of Chinese hamster ovary cells ( CHO-S) , and to verify the scavenging effect of rhα-Gal A on globular trisaccharide ceramide (Gb3 or GL3) . Methods The construction of recombinant protein expression vector, pcDNA4-GLA, was achieved by fusing the human α-galactosidase cDNA, gla, with 6 X His tag and artificial DNA synthesis. The expression plasmid was transfected into the suspended CHO-S to express rhα-Gal A and then purified. Following this procedure, we determined rhα-Gal A's expression, the enzymatic activity, and the glycosylation of the recombinant enzyme. Co-incubation with cultured cells was performed to examine whether rhα-Gal A could be taken up into the cells and effectively remove Gb3 substrates. Results rhα-Gal A was successfully expressed and purified after transiently transfecting pcDNA4-GLA into the suspended CHO-S, and the yield was up to (100 ±20. 6) mg • L
Texto completo:
DisponíveL
Índice:
WPRIM (Pacífico Ocidental)
Idioma:
Chinês
Revista:
Chinese Pharmacological Bulletin
Ano de publicação:
2023
Tipo de documento:
Artigo
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