Display of aggregation-prone ligand binding domain of human PPAR gamma on surface of bacteriophage lambda.
OBJECTIVE
To display the aggregation-prone ligand binding domain (LBD) of the human peroxisome proliferator-activated receptor gamma (PPARgamma) on the surface of bacteriophages to establish an easy screening assay for the identification of PPARgamma ligands.


METHODS
Plasmids were constructed for the expression of the PPARgamma 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.


CONCLUSIONS
The hydrophobic PPARgamma LBD was expressed as a soluble form of fusion protein in E coli and displayed on the surface of bacteriophage lambda when it was fused to the lambda pD protein. The lambda pD fusion system could be used for improving the solubility of proteins that tend to form inclusion bodies when expressed in E coli. The lambda phage particles displaying the LBD of PPARgamma may be of great value for the identification of novel PPARgamma ligands.
Kong
Bo
B
Institute of Health Sciences, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, and Medical School of Shanghai Jiao Tong University, China.
Ma
Wei-jun
WJ
D010582:Bacteriophage lambda / Q000235:genetics* / Q000378:metabolism; D001665:Binding Sites; D036022:Capsid Proteins; D004268:DNA-Binding Proteins / Q000096:biosynthesis* / Q000235:genetics; D004926:Escherichia coli / Q000378:metabolism*; D015870:Gene Expression; D008024:Ligands; D047495:PPAR gamma / Q000096:biosynthesis* / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics; D014760:Viral Fusion Proteins / Q000096:biosynthesis* / Q000235:genetics; D014764:Viral Proteins / Q000096:biosynthesis* / Q000235:genetics
D036022:Capsid Proteins; C041745:D protein, Enterobacteria phage lambda; D004268:DNA-Binding Proteins; D008024:Ligands; D047495:PPAR gamma; D011993:Recombinant Fusion Proteins; D014760:Viral Fusion Proteins; D014764:Viral Proteins