Immobilized fusion protein affinity chromatography combined with HPLC-ESI-Q-TOF-MS/MS for rapid screening of PPARγ ligands from natural products.
Screening agonists of peroxisome proliferator-activated receptor-γ (PPARγ) from natural products is particularly motivated by the need for effective anti-diabetic agents. However, method for direct identification of PPARγ ligands from a complex sample is rarely reported. Here we propose a novel immobilized fusion protein affinity chromatography (IFPAC) to achieve rapid multicomponent screening. First, functional human PPARγ ligand binding domain (hPPARγLBD) was bacterially produced by fusion to glutathione S-transferase (GST). The unpurified GST-hPPARγLBD was directly applied to a 96-well filter plate prepacked with glutathione sepharose. Due to the strong affinity between GST and glutathione, the fusion protein could selectively attach to the glutathione matrix with an oriented immobilization, which was rapidly purified under non-denaturing conditions. Experimental results indicated that the prepared 96-affinity column array exhibited excellent selectivity and sensitivity for fishing novel interacting compounds. The proposed approach was applied in the high-throughput screening of PPARγ ligands from natural products, followed by rapid characterization of active compounds using HPLC-ESI-Q-TOF-MS/MS. Isochlorogenic acid A in Dendranthema indicum flowers was found to be a PPARγ ligand. Our findings suggested the IFPAC could be a powerful tool for drug discovery from natural products.
College of Pharmaceutical Sciences, Zhejiang University, Hangzhou 310058, PR China.
Junfeng
J
Zhu
College of Pharmaceutical Sciences, Zhejiang University, Hangzhou 310058, PR China.
Xiaojiao
X
Yi
College of Pharmaceutical Sciences, Zhejiang University, Hangzhou 310058, PR China.
Wenhui
W
Liu
College of Pharmaceutical Sciences, Zhejiang University, Hangzhou 310058, PR China.
Yingchun
Y
Xu
College of Pharmaceutical Sciences, Zhejiang University, Hangzhou 310058, PR China.
Shuqing
S
Chen
College of Pharmaceutical Sciences, Zhejiang University, Hangzhou 310058, PR China. Electronic address: yjwu@zju.edu.cn.
Yongjiang
Y
Wu
D019659:Asteraceae / Q000378:metabolism; D002726:Chlorogenic Acid / Q000031:analogs & derivatives* / Q000032:analysis / Q000378:metabolism; D002846:Chromatography, Affinity / Q000379:methods*; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D055808:Drug Discovery / Q000379:methods*; D004800:Enzymes, Immobilized / Q000378:metabolism; D035264:Flowers / Q000378:metabolism; D005978:Glutathione / Q000378:metabolism; D005982:Glutathione Transferase / Q000378:metabolism; D008024:Ligands; D047495:PPAR gamma / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*; D053719:Tandem Mass Spectrometry / Q000379:methods*
D004800:Enzymes, Immobilized; D008024:Ligands; D047495:PPAR gamma; D011993:Recombinant Fusion Proteins; C469310:isochlorogenic acid; D002726:Chlorogenic Acid; D005982:Glutathione Transferase; D005978:Glutathione
HPLC–ESI-Q-TOF-MS/MS; Immobilized fusion protein affinity chromatography; Multicomponent screening; Natural products; Peroxisome proliferator-activated receptor-γ