重组人α-半乳糖苷酶在悬浮CHO-S细胞中的表达、纯化及功能活性鉴定

Expression, purification and functional verification of recombinant human α-galactosidase A in suspension CHO-S

目的 运用无血清可高密度悬浮培养的中国仓鼠卵巢细胞(Chinese hamster ovary cells, CHO-S)表达体系,分泌表达并纯化重组人α-半乳糖苷酶(recombinant human α-galactosidase A, rhα-Gal A),验证其对法布雷病贮积底物神经酰胺三己糖苷(globotriaosylceramide, Gb3 or GL3)的清除效果。方法 构建人α-半乳糖苷酶基因gla融合6×His标签的表达载体pcDNA4-GLA,将其转染至悬浮CHO-S细胞中,表达纯化并检测rhα-Gal A糖基化形式、表达量及酶活性;通过与人皮肤成纤维细胞共孵育,观察rhα-Gal A是否能被摄取到胞内并有效清除Gb3。结果 研究结果显示,rhα-Gal A成功在CHO-S细胞中表达,表达量最高可达(100±20.6) mg·L^(-1),且rhα-Gal A被糖基化修饰,酶活与商品酶Fabrazyme酶活相近,为(59±9.1) kU·g^(-1);此外,rhα-Gal A能被有效摄取到人成纤维细胞胞内并靶向溶酶体进而有效清除Gb3。结论 利用瞬时转染技术在悬浮CHO-S细胞中成功表达并纯化出rhα-Gal A,具有良好的生物学活性且可有效清除Gb3,为我国法布雷病的酶替代疗法提供新的选择。

Aim To express and purify rhα-Gal A with a 6×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 humanα-galactosidase cDNA,gla,with 6×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 -1 , and the expressed rhα-Gal A was glycosylated as expected. The enzymatic activity was similar to that of the commercial enzyme, Fabrazyme, which was (59±9.1) kU·g^(-1) . In addition, rhα-Gal A could effectively remove Gb3, be taken up by cultured human fibroblasts, and target to lysosomes. Conclusions The results of this study demonstrate successful high expression of the rhα-Gal A in suspension CHO-S by a transient transfection system. The recombinant enzyme has an excellent biological activity that matches Fabrazyme. We have provided a potentially new choice for enzyme replacement therapy for Fabry disease.