兼性离子修饰亲水磁珠固定化谷胱甘肽-S-转硫酶P1及其表征

Immobilization and characterization of glutathione-S-transthiase P1 on zwitterion-coated hydrophilic magnetic beads

谷胱甘肽-S-转硫酶P1(Human glutathione-S-transferaseπ1,h GSTP1)为肿瘤耐药增敏药靶。为探索磁珠固定化h GSTP1的合适方案用于磁分离指数富集从混合物中筛选高亲和力配体,重组表达6×His-h GSTP1,分别用Ni^2+-NTA琼脂糖和GSH-Sepharose 4B层析柱纯化,采用亲水兼性离子修饰的羧基磁珠和Ni^2+-NTA磁珠固定化6×His-h GSTP1,表征其固定化容量、保留活性、稳定性以及对抑制剂响应性。羧基磁珠不能有效固定GSH-Sepharose 4B层析柱纯化h GSTP1,可有效固定Ni^2+-NTA层析柱纯化h GSTP1,但最大保留酶活仅约30%。Ni^2+-NTA磁珠可有效固定两种方法纯化蛋白,其饱和固载容量约为羧基磁珠的1.7倍且最大保留酶活均可达80%。Ni^2+-NTA磁珠固定化h GSTP1在pH 6.5 PBS中0℃振摇8 h活性未见明显改变,相同条件游离酶活性下降约20%;在筛选后甲醇80℃处理20 min不脱落。Ni^2+-NTA磁珠固定化h GSTP1和游离酶对抑制剂依他尼酸(Ethacrynic acid,EA)的IC50无显著性差异(P>0.05)。因此,Ni^2+-NTA磁珠固定化h GSTP1可适用于指数富集筛选混合物库中抑制剂。

Human glutathione-S-transferase Pi isozyme(h GSTP1) is a target to tackle resistance of tumor. To explore an immobilization method suitable for affinity-drive-exponential-enrichment through magnetic separation for the screening of high-affinity ligands of h GSTP1 from mixtures, immobilizations of h GSTP1 on zwitterion-coated magnetic beads functionalized with Ni^2+-NTA and carboxyl were compared. The recombinant expressions of 6×his-h GSTP1 were purified by Ni^2+-NTA agarose column and GSHSepharose 4 B column, respectively. The immobilization capacity, retention activity, stability and response of immobilized h GSTP1 to inhibitor were characterized. Carboxyl-functionalized magnetic beads showed undetectable immobilization of h GSTP1 purified with GSH-Sepharose 4 B column and exhibited certain immobilization of h GSTP1 purified with Ni^2+-NTA agarose column, yielding about 30% retention activity of the free h GSTP1. While Ni^2+-NTA magnetic beads could effectively immobilize h GSTP1 purified with both columns, with a retention activity of about 80%, and its saturation immobilization capacity was 1.7 times that of carboxyl-functionalized magnetic beads. The immobilized h GSTP1 by Ni^2+-NTA magnetic beads was stirred in PBS(pH 6.5) at 0 ℃ for 8 h and did not show obvious change in activity, while the free enzyme activity decreased about 20%. After it was further treated with methanol at 80 ℃ for 20 min, negligible protein was released. There was no significant difference in IC50 of ethacrynic acid(EA) between h GSTP1 immobilized by Ni^2+-NTA magnetic beads and free enzyme(P>0.05). Therefore, the immobilized h GSTP1 by Ni^2+-NTA magnetic beads is suitable for affinity-drive-exponential-enrichment of h GSTP1 inhibitors in the mixture for screening.