A preparation strategy for protein-oriented immobilized silica magnetic beads with Spy chemistry for ligand fishing
- Author:
Yi YU
1
;
Hu JIANMING
;
Ding SHENWEI
;
Mei JIANFENG
;
Wang XUDONG
;
Zhang YANLU
;
Chen JIANSHU
;
Ying GUOQING
Author Information
1. Institute of Biopharmaceuticals,College of Pharmaceutical Science,Zhejiang University of Technology,Hangzhou,310014,China
- Keywords:
EDC/Sulfo-NHS;
SpyTag/SpyCatcher;
Green fluorescent protein;
Silica magnetic beads;
Oriented immobilization;
Drug screening
- From:
Journal of Pharmaceutical Analysis
2022;12(3):415-423
- CountryChina
- Language:Chinese
-
Abstract:
Due to the complexity of bioactive ingredients in biological samples,the screening of target proteins is a complex process.Herein,a feasible strategy for directing protein immobilization on silica magnetic beads for ligand fishing based on SpyTag/SpyCatcher(ST/SC)-mediated anchoring is presented.Carboxyl functional groups on the surface of silica-coated magnetic beads(SMBs)were coupled with SC using the 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride/N-hydroxysulfosuccinimide method,named SC-SMBs.The green fluorescent protein(GFP),as the capturing protein model,was ST-labeled and anchored at a specific orientation onto the surface of SC-SMBs directly from relevant cell lysates via ST/SC self-ligation.The characteristics of the SC-SMBs were studied via electron microscopy,energy dispersive spectroscopy,and Fourier transform infrared spectroscopy.The spontaneity and site-specificity of this unique reaction were confirmed via electrophoresis and fluorescence analyses.Although the alkaline stability of ST-GFP-ligated SC-SMBs was not ideal,the formed isopeptide bond was unbreakable under acidic conditions(0.05 M glycine-HCl buffer,pH 1-6)for 2 h,under 20％ethanol solution within 7 days,and at most temperatures.We,therefore,present a simple and universal strategy for the preparation of diverse protein-functionalized SMBs for ligand fishing,prompting its usage on drug screening and target finding.
