Citation: 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. doi: 10.11944/j.issn.1000-0518.2019.04.180269 shu

Preparation and Application of a Bivalent Affinity Labeling Reagent of Fusion Tag Glutathione S-Transferase

LU Jinpeng ^a ,
XIA Chao ^a ,
ZHOU Wei ^a ,
LI Yaping ^a ,
HU Xiaolei ^a ,
LIAO Fei ^b ,
YANG Xiaolan ^a,,

a.
Key Laboratory of Medical Laboratory Diagnostics of the Ministry of Education of China, College of Laboratory Medicine, Chongqing Medical University, Chongqing 400016, China
b.
School of Pharmacy and Bioengineering, Chongqing University of Technology, Chongqing 401135, China

Corresponding author: YANG Xiaolan, xiaolanyang666@yeah.net
Received Date: 17 August 2018
Revised Date: 22 November 2018
Accepted Date: 5 December 2018

Fund Project: Supported by the National Natural Science Foundation of China(No.31570862, No.81773625)National Natural Science Foundation of China 81773625National Natural Science Foundation of China 31570862

Figures(10)

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.
- glutathione S-transferase,
- affinity labeling reagents,
- magnetic particles,
- immobilization
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