Most protein-ligand interactions take place on surfaces and include but not limited to factors such as chemical composition, hydrophobicity, electronegavitiy and shape complementarity. Past studies showed that protein...Most protein-ligand interactions take place on surfaces and include but not limited to factors such as chemical composition, hydrophobicity, electronegavitiy and shape complementarity. Past studies showed that protein-protein interactions occur on comparatively fiat regions whereas protein-ligand bindings involve crevices. In the search for such sites various approaches have been designed and developed each of which is algorithmically unique. The use of grid units or voxels has been demonstrated in early studies with relatively good results obtained. We present here an approximated approach comprising of the use of voxels and computer vision methods in the search for ligand-binding areas. Each test protein is modelled and analysed in 2D with all corresponding residues graphically presented for successfully identified sites. The study was carried out on 2 sets of proteins： FK506-bound proteins and heme-bound proteins with promising results obtained for all test cases.展开更多
Peroxisome proliferator-activated receptors （PPARs） are ligand-activated transcription factors known to play a pivotal role in regulations of metabolism. In order to yield soluble ligand binding domain of PPARδ （P...Peroxisome proliferator-activated receptors （PPARs） are ligand-activated transcription factors known to play a pivotal role in regulations of metabolism. In order to yield soluble ligand binding domain of PPARδ （PPARδLBD） for screening ligands, the cDNA was amplified using total RNA from HepG2 cells by RT-PCR. Then the enzyme-digested product was inserted . downstream of the malE gene in the vectorpMAL-p2x, which encoded maltose-binding protein （MBP）, resulting in the expression of an MBP-PPARδLBD fusion protein. The recombinant plasmid was transformed into E. coli TBI that was cultured shakily at 30 ℃, 200 r/min and induced by 0.4 mmol/L IPTG for 6 h. The cells were harvested by centrifugation and broken by sonication. The expressed fusion protein was soluble and accounted for 0.31 of the total protein in the supernatant. Western blot analysis showed that the expressed MBP-PPARδLBD could bind to anti-MBP-antibody. The MBP-PPARδLBD fusion protein of 77 kDa and the PPARδLBD protein of 34 kDa were obtained by amylose-resin affinity chromatography without or with digestion of Factor Xa. They were both homogeneity, judged by SDS-PAGE. The recombinant MBP-PPARδLBD and PPARδLBD protein with high purity is obtained, which provides the necessary material for screening and researching PPARδ ligands.展开更多
文摘Most protein-ligand interactions take place on surfaces and include but not limited to factors such as chemical composition, hydrophobicity, electronegavitiy and shape complementarity. Past studies showed that protein-protein interactions occur on comparatively fiat regions whereas protein-ligand bindings involve crevices. In the search for such sites various approaches have been designed and developed each of which is algorithmically unique. The use of grid units or voxels has been demonstrated in early studies with relatively good results obtained. We present here an approximated approach comprising of the use of voxels and computer vision methods in the search for ligand-binding areas. Each test protein is modelled and analysed in 2D with all corresponding residues graphically presented for successfully identified sites. The study was carried out on 2 sets of proteins： FK506-bound proteins and heme-bound proteins with promising results obtained for all test cases.
基金Supported by the National Natural Science Foundation of China (30572353)
文摘Peroxisome proliferator-activated receptors （PPARs） are ligand-activated transcription factors known to play a pivotal role in regulations of metabolism. In order to yield soluble ligand binding domain of PPARδ （PPARδLBD） for screening ligands, the cDNA was amplified using total RNA from HepG2 cells by RT-PCR. Then the enzyme-digested product was inserted . downstream of the malE gene in the vectorpMAL-p2x, which encoded maltose-binding protein （MBP）, resulting in the expression of an MBP-PPARδLBD fusion protein. The recombinant plasmid was transformed into E. coli TBI that was cultured shakily at 30 ℃, 200 r/min and induced by 0.4 mmol/L IPTG for 6 h. The cells were harvested by centrifugation and broken by sonication. The expressed fusion protein was soluble and accounted for 0.31 of the total protein in the supernatant. Western blot analysis showed that the expressed MBP-PPARδLBD could bind to anti-MBP-antibody. The MBP-PPARδLBD fusion protein of 77 kDa and the PPARδLBD protein of 34 kDa were obtained by amylose-resin affinity chromatography without or with digestion of Factor Xa. They were both homogeneity, judged by SDS-PAGE. The recombinant MBP-PPARδLBD and PPARδLBD protein with high purity is obtained, which provides the necessary material for screening and researching PPARδ ligands.
