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Acta Pharmacologica Sinica 2006 January; 27 (1): 91-99; doi: 10.1111/j.1745-7254.2006.00253.x |
| Original Article | [ Full text ] |
| Display of aggregation-prone ligand binding domain of human PPAR gamma on surface of bacteriophage lambda1 |
Bo KONG2,4, Wei-jun MA2,3,5 2Institute of Health Sciences, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences and Medical School of Shanghai Jiao
Tong University, Shanghai 200025, China; 3Shanghai Research Center of Biotechnology, Shanghai Institutes for Biological Sciences,
Chinese Academy of Sciences, Shanghai 200233, China; 4Graduate School of the Chinese Academy of Sciences |
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Aim: To display the aggregation-prone ligand binding domain (LBD) of the human peroxisome proliferator-activated receptor gamma (PPARg) on the surface of bacteriophages to establish an easy screening assay for the identification of PPARg ligands.
Methods: Plasmids were constructed for the expression of the PPARg LBD as a fusion to the N-terminus of the g3p protein of filamentous phage or the C-terminus of the capsid protein D (pD) of phage lambda. The fusion proteins were expressed in E coli and solubility characteristics were compared. Polyclonal antibodies against the LBD as well as the pD protein were prepared for Western blot analysis and phage capture assay.
Results: The pD-LBD fusion protein was partially soluble, whereas the LBD-g3p fusion protein was detected only in the insoluble fraction. The pD-LBD fusion protein was efficiently incorporated in phage particles. Furthermore, the LBD was shown to be displayed on the surface of bacteriophage lambda. On average, the pD-LBD fusion protein accounted for 28% of the total pD protein in the lambda head capsid.
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Keywords: protein expression; lambda phage; surface display; nuclear receptor; peroxisome proliferator-activated receptors; ligand binding domain |
| 1 Project supported by the Major Fundamental Project of Shanghai Municipal Commission of Science and Technology (No 02DJ14007) and the Knowledge Innovation Program of the Chinese Academy of Sciences. |
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