Expression, purification and functional verification of recombinant human α-galactosidase A in suspension CHO-S
- Author:
Mu-Lan DENG
1
;
Hong-Yu ZHOU
1
;
Ke-Xin ZHENG
1
;
Zhao-Yang LI
1
;
Wan-Yi GUO
1
;
Yan-Ping WANG
1
;
Zhi-Cheng LIANG
1
;
Fang-Hong LI
1
;
Yun-Ping MU
1
;
Zi-Jian ZHAO
1
Author Information
- Publication Type:Journal Article
- Keywords: enzyme replacement therapy; fabry disease; genetic engineering; lysosomal storage disease; recombinant human α-galactosidase A; suspension CHO-S
- From: Chinese Pharmacological Bulletin 2023;39(4):774-781
- CountryChina
- Language:Chinese
- 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
