[Large recombinant protein displayed on filamentous phage surface and its interaction with small molecule].
Recombinant proteins were expressed as fusions with the phagemid system of pHEN-KM13 and the characteristics and activities of the fusion proteins displayed on the surface of filamentous phagintain the were studiedon abilty. The altered titer of rescued phages from the phagemid system after trypsin treatment indicated the relative quantity of the phages displaying fusion proteins. The rescue phages displayed foreign proteins could keep the bacterial infection ability, while the bald phage without foreign protein displayed on its surface was sensitive to trypsin treatment and lost the bacterial infection ability. To determine the upper limit for filamentous phage display, four recombinant proteins, glutathione-S-transferase and glutathione-S-transferase fused with three various size peptide linkers, were fused to N terminus of capsid protein gp3 and rescued by helper phage KM13. The rescued phages which displayed fused protein with the size of 40kD or less maintain the infection ability. To assay the activity of the phage displayed protein, the known small molecule probe was used in the interaction study with protein incorporated on phage surface. Results showed that the glutathione-S-transferase on phage surface still bound to glutathione specifically. It indicated that the glutathione-S-transferase displayed on phage surface was correctly folded and functionally active. The results demonstrated that it was feasible to use small molecule probes to interact with the protein displayed on phage surface. In turn, the method described here also demonstrated that phage display system could be utilized to investigate the interactions between protein and small molecules.
Institute of Health Sciences, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences & Medical School of Shanghai Jiao Tong University, 225 South Chongqing Road, Shanghai 200025, China.
Bo
B
Kong
Hui
H
Liu
Sheng-Li
SL
Yang
Wei-Jun
WJ
Ma
D005978:Glutathione / Q000235:genetics / Q000378:metabolism*; D005982:Glutathione Transferase / Q000096:biosynthesis / Q000235:genetics*; D006378:Helper Viruses / Q000235:genetics / Q000378:metabolism; D017912:Inovirus / Q000235:genetics / Q000378:metabolism*; D019151:Peptide Library; D011485:Protein Binding; D054730:Protein Interaction Domains and Motifs / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000235:genetics
D019151:Peptide Library; D011993:Recombinant Fusion Proteins; D005982:Glutathione Transferase; D005978:Glutathione