A general strategy for protein affinity-ligand oriented-immobilization and screening for bioactive compounds.

Natural products containing complex mixtures of potentially bioactive compounds are a major source of new drugs, however, conventional screening for active compounds is a time-consuming and inefficient process. Here, we reported that a facile and efficient protein affinity-ligand oriented-immobilization strategy based on the SpyTag/SpyCatcher(ST/SC) chemistry, was used for bioactive compound screening. Two ST-fused model proteins, that is, GFP (green fluorescent protein) and PqsA (a critical enzyme in the quorum sensing pathway of Pseudomonas aeruginosa), were used to verify the feasibility of this screening method. GFP, as the capturing protein model, was ST-labeled and anchored at a specific orientation onto the surface of activated agarose coupled with SC protein via ST/SC self-ligation. The affinity carriers were characterized by infrared spectroscopy and fluorography. The spontaneity and site-specificity of this unique reaction were confirmed via electrophoresis and fluorescence analyses. Although the alkaline stability of the affinity carriers was not ideal, its pH stability was acceptable under pH < 9. The general preparation strategy of this affinity carriers was validated by replacing GFP with PqsA, and PqsA inhibitor, 2-amino-6-fluorobenzoic acid, was successfully isolated from the fermentation broth. The proposed strategy can immobilize protein ligands in one-step and screen compounds that interact specifically with the ligands.

A preparation strategy for protein-oriented immobilized silica magnetic beads with Spy chemistry for ligand fishing.

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.

A preparation strategy for protein-oriented immobilized silica magnetic beads with Spy chemistry for ligand fishing / 药物分析学报

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.