标签谷胱甘肽S-转移酶的二价亲和标记试剂的制备与应用

doi:10.11944/j.issn.1000-0518.2019.04.180269

摘要/Abstract

摘要：

设计合成融合表达标签谷胱甘肽S-转移酶(GST)的二价亲和标记试剂,用于功能化磁珠后位点选择性固定化标签GST,为磁分离筛选配体混合物库提供固定化融合靶蛋白的候选方案。为减少疏水配体在标签GST活性位点的结合,需同时占据标签GST双活性中心内疏水结合位点并发生共价修饰的二价亲和标记试剂。以双苯环为疏水定位基、溴乙酰基为巯基修饰基团、羧基为连接官能团得单价标记试剂,以二乙基三胺为连接臂将单价标记试剂与连接臂两端伯胺连接得标签GST的对称二价亲和标记试剂,再以线性三胺连接臂中间的氨基与羧基磁珠偶联得功能化磁珠。表征目标化合物对标签GST的标记动力学、结合比;功能化磁珠对标签GST的不可逆固定化动力学和固载容量,及将磁珠表面二价亲和标记试剂转变成还原型谷胱甘肽(GSH)加合物后对标签GST可逆固定化的效果;以碱性磷酸酶及疏水荧光配体为模型考察磁珠固定化标签GST后的非特异结合。目标化合物对标签GST半抑制浓度为(22±0.2) μmol/L,其与GSH的饱和加合物半抑制浓度为(0.41±0.06) μmol/L,二者与标签GST二聚体结合比接近1：1。功能化磁珠对标签GST不可逆及可逆固定化的容量均接近25 mg/g磁珠。偶联GST的磁珠对蛋白非特异吸附很弱,再进一步用单价亲和标记试剂和GSH加合物封闭固定化标签GST剩余的活性位点后对疏水小分子也无显著结合。结果表明,所设计二价亲和标记试剂功能化磁珠适合用于标签GST及其融合表达蛋白的位点选择性固定化。

关键词: 谷胱甘肽S-转移酶, 亲和标记试剂, 磁珠, 固定化

Abstract:

To develop a platform for the site-selective immobilization of proteins and the screening of mixture-based ligand libraries through magnetic separation of complexes of ligands and targets, a bivalent affinity labeling reagent(Br-Ⅱ) against fusion tag glutathione S-transferase(GST) was designed and characterized. The use of two flexible benzenes as the moiety to occupy the hydrophobic site in one active site of GST, bromoacetyl as the group for covalent modification of sulfhydryl group nearby and carboxyl as the linking group yielded a monovalent affinity labeling reagent; the linkage of the monovalent affinity labeling reagent to the primary amines on diethylenetriamine gave Br-Ⅱ as the bivalent affinity labeling reagent that contained a secondary amino group for further conjugation with carboxyl group on magnetic beads. The kinetics and ratios for irreversible and reversible labeling of GST tag, before and after conjugation with reduced glutathione(GSH), respectively, was characterized for the designed bivalent affinity labeling reagent. Using alkaline phosphatase as the unwanted protein and the fluorescent ligand 8-anilino-1-naphthalenesulfonic acid as the small molecule model, the immobilization specificity for GST tag, and also the nonspecific adsorption for hydrophobic small molecules of the resultant GST-immobilized magnetic beads were checked. The designed compound showed half inhibitory concentration of (22±0.2) μmol/L, and its GSH adduct gave half inhibitory concentration of (0.41±0.06) μmol/L; the reversible and irreversible labeling of GST tag dimer showed both binding ratios close to 1：1; the Br-Ⅱ-functionalized magnetic beads showed selective immobilization of GST tag in the presence of alkaline phosphatase, and the immobilization capacity of about 25 mg/g for GST via either reversible or irreversible immobilization. After the immobilization of GST and further treatment with the GSH adduct of the monovalent affinity labeling reagent, magnetic beads showed no significant nonspecific binding of 8-anilino-1-naphthalenesulfonic acid. Therefore, magnetic beads after being functionalized with the titled compound are suitable for the immobilization of GST-fused proteins.

Key words: glutathione S-transferase, affinity labeling reagents, magnetic particles, immobilization

卢进鹏, 夏超, 周伟, 李亚平, 胡小蕾, 廖飞, 杨晓兰. 标签谷胱甘肽S-转移酶的二价亲和标记试剂的制备与应用[J]. 应用化学, 2019, 36(4): 402-413.

LU Jinpeng, XIA Chao, ZHOU Wei, LI Yaping, HU Xiaolei, LIAO Fei, YANG Xiaolan. Preparation and Application of a Bivalent Affinity Labeling Reagent of Fusion Tag Glutathione S-Transferase[J]. Chinese Journal of Applied Chemistry, 2019, 36(4): 402-413.

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